siRNA细胞转染实验步骤(Entranster)

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siRNA细胞转染实验操作指南

siRNA细胞转染实验操作指南siRNA产品的最佳工作浓度、转染后检测时间因不同的细胞类型和研究目的而异。

一般推荐的siRNA工作浓度为50nM，检测时间为转染后24~72h。

可以通过设置时间梯度和浓度梯度进行组合实验来选择最优的siRNA工作浓度和检测时间。

由于siRNA过量会对细胞产生毒性，实验建议的siRNA 工作浓度优化的范围为5~100nM。

下面以Lipofectamine 2000转染24孔板的293T细胞为例，选取50nM的siRNA工作浓度，介绍siRNA转染细胞的操作步骤。

若使用其他的转染试剂，请参照对应的产品说明书进行操作。

1.在转染前一天，按1×105~5×105个/孔的量将细胞接种于不含抗生素的完全培养基中，使次日转染时细胞融合度30-50%为宜。

接种时尽量保证每孔细胞的接种数量一致，使细胞均匀地平铺在生长表面。

注意:降低转染时的细胞密度可延长转染和收样的时间间隔，避免细胞过度生长对实验结果造成影响。

可根据细胞特性和实验目的等，调整接种时的细胞密度。

2.每个待转染的细胞样品（每个培养孔），按以下体系配置转染所需的siRNA-Lipofectamine 2000复合物:（1）配制稀释液A:取25pmol siRNA于50ul Opti-MEM无血清培养基中稀释，轻轻混匀。

（*siRNA的用量计算参照表1）（2）配制稀释液B:使用前先将Lipofectamine 2000轻轻混匀，取出1ul于50ul Opti-MEM无血清培养基中稀释，轻轻混匀，室温下孵育5分钟。

注意孵育时间不能超过25min。

（3）孵育完成后，将稀释液A与B轻轻混匀得到siRNA-Lipofectamine 2000复合物，在室温下孵育20分钟。

此时溶液可能会浑浊，属于正常现象。

表1 不同细胞培养板siRNA 转染用量参考表3.将孵育好的siRNA-Lipofectamine 2000复合物分别加到对应的细胞孔中，轻轻混匀。

siRNA干扰实验步骤(贴壁细胞)

1．转染前一天，4-5×10* 4细胞接种在24 孔板上，0.5 含和抗生素的（或，其他培养基）细胞培养基。

2．选择用于初期接种的细胞数量，应能在24小时内使细胞汇合达到70-90%。

3. 在50μl的（或，或其他无血清培养基）无血清培养基加入20 (或0.8μg )，柔和混匀；
4. 混匀试剂，用50μl 无血清的或，或其他无血清培养基）稀释1μl 试剂（转染时，则加入2μ试剂），轻轻混匀，室温放置5分钟；
5. 将稀释好的和试剂混合；轻柔混匀，室温放置20 分钟, 以便形成（或）复合物。

6. 将100μl （或）复合物加到含有细胞和培养基的培养板的孔中，来回轻柔摇晃细胞培养板板。

7．细胞在2培养箱中37℃温育2448h后，进行转染后的其它检测步骤。

如果细胞株比较敏感，孵育4-6小时后，除去复合物，更换培养基。

siRNA转染(Entranster)研究成纤维细胞瘤,转染效率高

ORIGINAL PAPERRIP1-dependent Bid cleavage mediates TNF a -inducedbut Caspase-3-independent cell death in L929ﬁbroblastoma cellsGuozhu Chen •Xiang Cheng •Ming Zhao •Song Lin •Jiangyang Lu •Jiarui Kang •Xiaodan YuÓSpringer Science+Business Media New York 2014Abstract L929ﬁbroblastoma cells (L929-A)and L929ﬁbrosarcoma cells (L929-N)are different cell lines that are commonly used to study the cytotoxicity of tumor necrosis factor alpha (TNF a ).TNF a has been reported to induce necrosis in both of these cell lines.However,comparing the TNF a -induced cell death in these two cell lines,we found that,unlike the L929-N cells that show typical RIP3-dependent necrosis,TNF a -induced cell death in L929-A cells is pan-caspase inhibitor Z-VAD-FMK (Z-VAD)-sen-sitive,which does not depend on RIP3.We also conﬁrmed that the cell death signal in the L929-A cells was initiated through cytosol-preassembled ripoptosome and that the knockdown of either Caspase-8or RIP1protein blocked cell pared with the L929-N cells,the L929-A cell line had lower levels of constitutive and inducible TNF a autocrine production,and the pan-caspase inhibitors Z-VAD or Q-VD did not kill the L929-A cells as they affect the L929-N cells.Moreover,the L929-A cells expressed less RIP3protein than the L929-N cells;there-fore,TNF a failed to induce RIP3-dependent necroptosis.In addition,the ripoptosome-mediated cell death signal was transduced to the mitochondria through Caspase-8-medi-ated and RIP1kinase activity-dependent Bid cleavage.The RIP1kinase inhibitor Necrostatin-1(Nec-1)or Caspase-8knockdown completely blocked Bid cleavage,and the knockdown of Bid or Bax/Bak prevented TNF a -induced cell death in the L929-A cells.Although the activation of Bax/Bak decreased the mitochondrial membrane potential,the levels of mitochondrial intermembrane space proteins,including cytochrome-c (cyt-C)and Smac,declined,and western blotting and immunoﬂuorescence staining analysis did not determine whether these proteins were redistributed to the cytosol.In addition,the mitochondrial outer mem-brane protein Tom20was also reduced,indicating that the reduced mitochondria proteins may be induced by the reduced mitochondria numbers.No efﬁcient cyt-C release was observed;therefore,the limited activation and cleav-age of downstream caspases,including Caspase-9,Cas-pase-7,Caspase-6and Caspase-3,was insufﬁcient to kill the cells.The Caspase-9,Caspase-6and Caspase-3/7inhibitors or Caspase-9and -3knockdown also failed to block cell death,and the overexpression of Bcl-2also did not abrogate cell death.Moreover,the dead cells showed necrotic-like but not apoptotic characteristics under transmission electronmicroscopy,and these features were signiﬁcantly different from mitochondrial apoptosis,indicating that the effector caspases were not the execu-tioners of cell death.These new discoveries show that TNF a -induced cell death in L929-A cells is different than typical RIP3-dependent necrosis and Caspase-8/Caspase-3-mediated apoptosis.These results highlight that caution is necessary when using different L929cells as a model to investigate TNF a -induced cell death.Keywords L929ﬁbroblastoma cells ÁL929ﬁbrosarcoma cells ÁTNF a ÁRIP1ÁRIP3ÁNecrosis ÁBid-cleavage Abbrevations TNF a Tumor necrosis factor alphaGuozhu Chen and Xiang Cheng have contributed equally to this work.G.Chen ÁX.Cheng ÁM.Zhao ÁS.Lin ÁX.Yu (&)Department of Cognitive Science,Institute of Basic Medical Sciences,Cognitive and Mental Health Research Center,#27Taiping Road,Beijing 100850,China e-mail:yuxd@J.Lu ÁJ.KangDepartment of Pathology,The First Afﬁliate Hospital of PLA,Beijing,ChinaApoptosisDOI 10.1007/s10495-014-1058-0Z-VAD Z-VAD-FMKNec-1Necrostatin-1MMP Mitochondrial membrane potentialROS Reactive oxygen speciescyt-C Cytochrome-cIntroductionProgrammed cell death is essential to the normal functions of multi-cellular organisms and plays a critical role in development,immunity,inﬂammation,and cancer pro-gression.Based on morphological and biochemical fea-tures,programmed cell death is classiﬁed into apoptosis, necrosis(also termed necroptosis),and autophagic cell death[1,2].Although many of the initiators,regulators, and effectors in these three types of cell death are different, increasing evidence indicates that apoptosis,necrosis,and autophagic cell death are not completely independent cell death pathways and that the various death pathways are closely intertwined with crosstalk between the regulating components.TNF a is a pleiotropic cytokine that induces a variety of cellular responses,including the promotion of cell survival and induction of programmed cell death.In addition to inducing Caspase-dependent apoptosis,TNF a also induces RIP1and RIP3kinase-dependent necroptosis as an alter-native during apoptosis-deﬁcient conditions[3,4].The mechanisms by which TNF a induces either apoptosis or necroptosis have been widely studied in recent years,and the cell death type is mediated by the formation of apop-totic or necrotic signaling complexes[5–7].Apoptosis is mediated by complex IIa,which contains RIP1,FADD, Caspase-8,and TRADD.In complex IIa,Caspase-8 becomes activated and then activates downstream Caspase-3to induce apoptosis[8–10].In certain cell lines,activated Caspase-8also induces apoptosis by cleaving Bid,which then activates the Bax/Bak pore and subsequent events, causing the activation of Caspases-9and-3[11,12].In cells with high RIP3expression and treatment with the broad-spectrum Caspase-inhibitor Z-VAD to inhibit the Caspase-8-induced cleavage of RIP1,RIP3,and CYLD, RIP3interacts with RIP1to form complex IIb(also known as the necrosome),which then initiates necroptosis[13–16]. Notably,in addition to death receptor activation-induced necrosis,many other inducers such as ischemia–reperfu-sion,oxidative stress,calcium overload,and others also induce necrosis[17].However,the mechanisms by which these intrinsic stimuli induce necroptosis remain primarily undeﬁned.Similar to TNF a inducing complex IIa forma-tion,genotoxic stress and the loss of IAPs are known to induce ripoptosome formation.Although the ripoptosome comprises the same components as complex IIa,the rip-optosome pre-assembles or spontaneously assembles and does not require pre-existing TNFR signaling platforms.In addition,the ripoptosome,in the absence or presence of RIP3,can either lead to RIP1kinase-dependent apoptosis or RIP1kinase-dependent necrosis,respectively[18,19]. In addition to inﬂuencing inducible ripoptosome assembly, whether RIP1kinase also involves the regulation of mito-chondria dysfunction and downstream effector caspase activation during ripoptosome-initiated apoptosis is cur-rently unknown.L929cells are commonly used to test the cytotoxic effects of TNF a;however,this short name actually corre-sponds to two different cell lines(L-929ﬁbroblastoma cells ATCCÒCCL-1TM and WEHI-13VARﬁbrosarcoma cells ATCCÒCRL-2148TM)is prone to cause some con-fusion.The common feature of these two L929cell lines is that both are sensitive to TNF a,and TNF a alone induces cell death.Although several early studies have reported Fig.1Z-VAD differentially regulates TNF a-induced cell death in L929-A and L929-N cells.a Nec-1inhibited TNF a-induced cell death in both L929-A and L929-N cells.The cells were treated with TNF a (10ng/mL for L929-A cells and50ng/mL for L929-N cells in all experiments)in the absence or presence of Nec-1(50l M in all experiments)for24h,and cell death was examined by microscopy (9200)as based on morphological changes.More than threeﬁelds in each group were observed,and representative images are shown.The cells were also stained with Annexin V-FITC and PI and analyzed by ﬂow cytometry.Representative measurements of at least three independent experiments are shown.The cell death values reported represent the mean±SD of three separate experiments.*P\0.01.b TNF a-induced cytotoxicity was inhibited in L929-A cells but promoted in L929-N cells in response to Z-VAD treatment.The cells were treated with TNF a in the absence or presence of Z-VAD(20l M in all experiments)for24h,and cell death was examined by morphological changes via microscopy(9200)andﬂow cytome-try.*P\0.01,#P\0.05.c Z-VAD and Nec-1suppressed the TNF a-induced cleavage of PARP and Caspase-3.The cells were treated with TNF a in the absence or presence of Z-VAD and Nec-1for12h. Western blotting was used to detect the cleavage of PARP and Caspase-3induced by TNF a.d Pan-caspase inhibitors induced cell death in L929-N cells but not L929-A cells.The cells were treated with Z-VAD or Q-VD(50l M)for48h.Cell death was examined by microscopy(9200)as based on morphological changes.The cells were also stained with propidium iodide and analyzed byﬂow cytometry.*P\0.01.e Z-VAD signiﬁcantly promoted TNF a tran-scription in L929-N cells but not L929-A cells.The cells were treated with Z-VAD for12h and then collected for RNA extraction.The TNF a mRNA level was determined by RT-PCR.The representative images of three independent experiments are shown.f The effect of Z-VAD on the activation of Erk in L929-A and L929-N cells.The cells were treated with20l M Z-VAD for the indicated time points and then lysed.The phosphorylated and total Erk levels were determined by western Blotting.g RIP3was differentially expressed in the L929-A and L929-N cells.The L929-A and L929-N cells were treated with TNF a for12h,and western blotting was used to determine the RIP1and RIP3levels.GAPDH was used as a loading controlcApoptosisthat TNF a induces apoptosis or atypical apoptosis in L929ﬁbroblastoma cells(for clariﬁcation we refer to this cell line here as L929-A)[20–22],a recent study demonstrated that TNF a induces necrotic cell death[23].In contrast, TNF a induces typical necroptosis in the L929ﬁbrosarcoma cell line(L929-N)[24–26].Although L929cells have been widely used as a model to study TNF a-induced necropto-sis,the results obtained from different studies involving the regulation of TNF a-induced necrosis are surprisingly var-iable or even contradictory.For example,RIP1knockdown has been reported to either enhance or inhibit the resistance of L929cells to TNF a[1,27],and autophagy initiation may either block necrosis or enhance necrosis[28,29].No explanations currently exist clarifying these differences; however,the respective L929cells used in these studies may have actually been different variants of this cell line. In this study,we compared the TNF a-induced cells death in these two L929cell lines,and unlike L929-N cells, TNF a-induced cell death in the L929-A cells was not RIP3-dependent necrosis but was RIP1-and Caspase-8-dependent and Caspase-3-independent cell death with necrotic morphology.These results indicate that we needto Apoptosisbe cautious when using different L929cells as a model to investigate TNF a-induced cell death.ResultsZ-VAD inhibits TNF a-induced cell death in L929-A cells but enhances death in L929-N cells via autocrine TNF a productionFirst,we treated L929-A and L929-N cells with the TNF a and RIP1kinase inhibitor,Nec-1,or the pan-caspase-inhibitor,Z-VAD,to observe their effects on cell death as evaluated either using microscopy orﬂow cytometry ana-lysis.Both cell lines were sensitive to TNF a,with the L929-A cells being more sensitive than the L929-N cells. Nec-1blocked TNF a-induced cell death in both cell lines, indicating that the cell death was RIP1-dependent(Fig.1a).However,the effects of Z-VAD on TNF a-induced cell death were contrasting.Speciﬁcally,Z-VAD exacerbated TNF a-induced cell death in the L929-N cells as previously reported[30],whereas Z-VAD inhibited cell death in the L929-A cells(Fig.1b).Western blot analysis also showed the cleaved Caspase-3and PARP levels in the L929-A cells treated with TNF a but not in the L929-N cells.More importantly,like Z-VAD,Nec-1also blocked the cleavage of Caspase-3and PARP(Fig.1c).A previous study reported that Z-VAD alone induces necroptosis in L929cells through the activation of MAPKs and AP-1and subsequent autocrine TNF a production[31]. We treated the cells with pan-caspase-inhibitors and found that both Z-VAD and Q-VD induce cell death in the L929-N cells but not in the L929-A cells(Fig.1d).We then assessed the effect of Z-VAD on TNF a transcription by RT-PCR.The results showed that L929-N cells constitu-tively expressed a high level of TNF a and that TNF a mRNA levels remarkably enhanced following Z-VAD stimulation,whereas the L929-A cells showed very low levels of constitutive TNF a expression,which were only faintly enhanced by Z-VAD(Fig.1e).In addition,we found that the L929-N cells have high levels of constitutive ERK activation(Fig.1f).Theseﬁndings support the hypothesis that the absence of autocrine TNF a in L929-A cells may allow the cells to escape Z-VAD-induced necrosis.A recent study showed that protein kinase RIPK3 activity determines whether cells die by necroptosis or apoptosis[32];therefore,we evaluated the expression of RIP1and RIP3proteins in these two L929cell lines fol-lowing TNF a exposure,and the expression of RIP3in the L929-N cells was much higher than that in L929-A cells. Moreover,TNF a reduced RIP1protein levels in both cell lines;however,in the L929-A cells,the RIP3protein levels were reduced at greater amounts compared with in the levels in the L929-N cells(Fig.1g).TNF a-induced L929-A cell death is RIP1-and Caspase-8-dependent but RIP3-independentThe key step for TNF a-induced apoptosis is the formation of complex IIa;therefore,weﬁrst investigated the associ-ation between RIP1,Caspase-8,and FADD in L929-A cells via immunoprecipitation analysis.Unexpectedly,a pre-existing interaction between the RIP1,Caspase-8,and FADD proteins was observed prior to TNF a stimulation, which is consistent with an assembled ripoptosome[19]; moreover,TNF a signiﬁcantly decreased the protein level of RIP1and Caspase-8in the whole cell lysate,causing less RIP1to be immunoprecipitated compared with the control. However,a higher level of Caspase-8was co-immuno-precipitated with RIP1,indicating that the association between RIP1and Caspase-8was remarkably enhanced in TNF a-treated cells(Fig.2a).Although TNF a induced time-dependent cell death,cleaved PARP did not exhibit the same increase,and the Caspase-8,RIP1,and cFLIP L proteins decreased(Fig.2b).The Caspase-8decrease was reversed by Nec-1and partially recovered by Z-VAD treatment but not by treatment with the Caspase-8inhibitor Z-IETD(Fig.2c).The effects of the components of com-plex II on TNF a-induced cell death were further investi-gated via the separate knockdown of Caspase-8,RIP1,and RIP3.The results conﬁrmed that TNF a-induced cell death in the L929-N cells occurred through RIP3-dependent necrosis(Fig.2d).In the L929-A cells,only the knock-down of RIP1or Caspase-8,not RIP3,markedly decreased the cytotoxicity of TNF a(Fig.2e).Fig.2Effect of TNF a on the composition of complex II proteins inL929-A cells.a TNF a promoted the binding between Caspase-8and RIP1.The cells were treated with or without TNF a for12h.An anti-RIP1antibody was used for immunoprecipitation,and western blotting was then used to analyze the binding between RIP1and Caspase-8.b The effects of TNF a on inducing time-dependent cell death and protein expression.The cells were treated with TNF a for the indicated times,cell death was examined by microscopy(9200), and the protein levels of FLIP L,RIP1,Caspase-8,and PARP were detect by western blotting.Actin was used as a loading control.c Nec-1and Z-VAD reversed the Caspase-8downregulation induced by TNF a.The cells were treated with TNF a in the absence and presence of Nec-1or Z-VAD for24h,and the Caspase-8protein level was detected by western blotting.d,e RIP1,RIP3,and Caspase-8 differentially regulated cell death induced by TNF a in the L929-N and L929-A cells.The cells were transfected with RIP1,RIP3,or Caspase-8siRNA for48h and then treated with TNF a for24h.Cell death was examined by microscopy(9200)and analyzed byﬂow cytometry.The effect of siRNA knockdown was determined by western blotting.*P\0.01cApoptosisApoptosisBid cleavage is important for TNF a-induced cell deathin L929-A cellsAs a BH3-only family member,Bid has been shown to play a crucial role in death receptor-induced apoptosis in certain type II cells[11,12].In addition to transferring the extrinsic apoptotic signal from the cytosol to the mitochondria,Bid cleavage also ampliﬁes the caspase cascade via the active intrinsic apoptosis pathway[11,12,33].We isolated mito-chondria and detected the Bid protein by western blotting.As shown in Fig.3a,full-length Bid was found in the mito-chondria and cytosol of the L929-A cells,and TNF a resulted in signiﬁcant Bid cleavage,which was only detected in iso-lated mitochondria(Fig.3a).Furthermore,the levels of several mitochondrial proteins,including cyt-C,AIF,and Smac,were also decreased concomitant with Bid cleavage (Fig.3a).TNF a-induced Bid cleavage and cyt-C loss were partially inhibited by Z-VAD(Fig.3b),whereas the Cas-pase-8inhibitor Z-IETD did not inhibit Bid cleavage or reverse cyt-C loss(Fig.3b).However,siRNA targeting Caspase-8completely blocked Bid cleavage and reversed AIF and cyt-C decline(Fig.3c),indicating that Caspase-8is involved in mediating Bid pared withZ-VAD,Nec-1completely blocked Bid cleavage and reversed cyt-C decrease,indicating that TNF a-induced Bid cleavage is RIP1kinase activity-dependent(Fig.3d).The importance of Bid in mediating TNF a-induced cell death was further conﬁrmed by the knockdown of Bid expression, which promoted cell death in the L929-N cells but not in the L929-A cells(Fig.3e).Effect of other proteases on Bid cleavageIn addition to Caspase-8,TNF a-induced JNK activation has been reported to induce the cleavage of Bid and pro-duce jBid,which functions similarly to tBid to promote apoptosis[34].TNF a stimulation leads to the accumulation of reactive oxygen species(ROS),an essential step for prolonged JNK activation and the induction of cell apop-tosis or necrosis[35,36].We also detected strong TNF a-induced sustained JNK activation in the L929-A cells,and the JNK inhibitor SP600125partially blocked TNF a-induced cell death;however,inhibiting JNK activity did not remarkably decrease Bid cleavage(Fig.4a).Further-more,the ROS scavengers affected cell viability and Bid cleavage in TNF-treated L929-A cells.Interestingly,these two ROS scavengers,butylated hydroxyanisole(BHA)and N-acetylcysteine(NAC),showed different effects.BHA effectively blocked TNF a-induced Bid cleavage and pro-tected the cell from TNF a-induced cell death,whereas NAC was not able to block Bid cleavage or prevent cell death(Fig.4b,c).Recently,Cabon et al.reported that alkylating DNA-damage agents mediate necroptosis,a process that involves calpain-mediated Bid cleavage[37].Thus,we treated the L929-A cells with the calpain inhibitors E-64-c and ALLN and found that these agents did not block TNF a-induced cell death(Fig.4d,e),and E-64-c did not inhibit Bid cleavage (Fig.4d).Similar to calpain,the possibility that lysosomal cathepsins and chymotrypin B mediate Bid cleavage[38,39] was also excluded because E-64-c also inhibited cathepsin B, cathepsin H,and cathepsin L.In addition,the cathepsin D inhibitor pepstatin A and chymotrypsin B inhibitor N-p-tosyl-L-phenylalaninechloromethylketone(TPCK)also failed to rescue the L929-A cells from TNF a-induced cell death (Fig.4e).Bid cleavage induces mitochondrial dysfunctionUsing a mitochondrial membrane potential assay kit with JC-1,we detected changes in the mitochondrial membrane potential(MMP).TNF a caused a signiﬁcant loss of MMP, whereas Z-VAD,Nec-1,and BHA signiﬁcantly rescued the loss of MMP(Fig.5a),which are consistent with the blockage of Bid cleavage by these molecules,indirectly conﬁrming that Bid cleavage mediates MMP loss.Cleaved Bid was previously demonstrated to induce a downstream cell death signal by activating Bax and Bak[40,41],and the knockdown of both Bax and Bak(but not Bax or Bik alone)also effectively inhibited TNF a-induced cell death (Fig.5b).We also detected the effects of other Bcl-2 family proteins on TNF a-induced cell death.The results Fig.3Bid cleavage is involved in mediating TNF a-induced necrop-tosis in L929-A cells.a TNF a induced Bid cleavage in L929-A cells. The cells were treated with or without TNF a for12h,the mitochondria and cytoplasm were separated,and the proteins were extracted.Western blotting was used to detect AIF,cyt-C,Smac and Bid cleavage.b Z-VAD but not Z-IETD partially reversed the Bid cleavage induced by TNF a.The cells were treated with TNF a in the absence or presence of Z-VAD or Z-IETD(50l M)for12h,then the mitochondria and cytoplasm were separated,and the proteins were extracted.Western blotting was used to detect Tubulin,Actin,cyt-C, and Bid cleavage.c Caspase-8knockdown reversed Bid cleavage induced by TNF a.The cells were infected with Caspase-8or control shRNA lentivirus for72h and then treated with or without TNF a for 12h.The mitochondria and cytoplasm were separated,and the proteins were extracted.Western blotting was used to detect Tubulin, COX IV,cyt-C,AIF,Caspase-8and the cleavage of Bid.d Nec-1 signiﬁcantly inhibited TNF a-induced Bid cleavage in L929cells.The L929-A cells were treated with TNF a in the presence or absence of Nec-1for12h.The mitochondria and cytoplasm were separated,and the proteins were extracted.Western blotting was used to detect Tubulin,Actin,cyt-C,and Bid cleavage.e Bid knockdown decreased L929-A cell death induced by TNF a.The cells were transfected with Bid siRNA or control siRNA for48h and then treated with or without TNF a for24h.Cell death was examined by microscopy(9200)and analyzed byﬂow cytometry.The effect of siRNA knockdown was determined by western blotting.*P\0.01cApoptosisApoptosisshowed that the transient overexpression of Bcl-xL par-tially decreased TNF a-induced cell death,but the overex-pression of Bcl-2had no protective effect(Fig.5c).The western blotting results showed that following Bid cleav-age,the mitochondrial intramembrane space proteins cyt-C and Smac were reduced,and these proteins did not appear in the ing MitoTracker to co-stain the cells,the distribution of cyt-C and AIF proteins in the cells was studied.The cyt-C protein did not show a remarkable release from mitochondria;however,the AIF protein was re-distributed in the nucleus following TNF a exposure (Fig.5d).Western blot analysis also showed increased AIF protein in the nucleoprotein fraction(Fig.5d),proving that the AIF protein entered the nucleus following TNF a exposure.Then,we detected the level of the mitochondria outer membrane protein Tom20,and TNF a reduced the levels of both cyt-C and Tom20proteins,indicating that the decline of mitochondrial intramembrane space proteins may be related to the decreasing number of mitochondria in the cells(Fig.5e).The effect of inhibiting caspase activation on TNF a-induced L929-A cell deathBid cleavage induces Bax/Bak-dependent cell death,but no obvious mitochondrial cyt-C release was observed;there-fore,we next investigated whether Bax/Bak activation was accompanied with apical Caspase-8and downstream Cas-pase-9and Caspase-3/7or Caspsase-6activation similar to typical apoptosis.First,western blot analysis of Caspase-activation in L929-A cells treated with TNF a showed that TNF a only induced faint cleavage of Caspase-8,Caspase-9,Caspase-7and Caspase-3,and Caspase-6did not show any cleavage.The cleaved Caspase-s and PARP did not show a time-dependent increase,which was inconsistent with time-dependent cell death(Figs.6a,2b).We then used several Caspase-inhibitors,including the pan-Cas-pase-inhibitor Q-VD,Caspase-8inhibitor Z-IETD,Cas-pase-9inhibitor Z-LEHD,Caspase-6inhibitor Z-VEID and Caspase-3/7inhibitor Z-DEVD,to separately pretreat L929-A cells and then stimulated with TNF a for24h. Only Q-VD prevented TNF a-induced cell death,whereas Z-IETD,Z-LEHD,Z-VEID and Z-DEVD had no signiﬁ-cant effects on TNF a-induced cell death(Fig.6b),and the knockdown Caspase-9did not affect TNF a-induced cell death(Fig.6b).The efﬁciency of these caspase inhibitors was demonstrated by using etopside-induced Jurkat cell apoptosis as a positive control(Fig.6c),and western blot analysis showed that Z-IETD and Z-DEVD block Caspase-8and Caspase-3/7cleavage separately in Jurkat cells but failed to block this cleavage in L929-A cells(Fig.6d).To further evaluate the effect of Caspase-3,we used siRNA to knockdown Caspase-3and further conﬁrmed that Caspase-3knockdown did not inhibit TNF a-induced cell death (Fig.6e)or affect TNF a-induced PARP cleavage(Fig.6f). In addition,cell morphological changes were observed by transmission electron microscopy.The results showed that TNF a exposure did not induce the characteristic apoptotic nuclear morphological features such as chromatin con-densation and the formation of apoptotic bodies;however, massive cytosolic vacuolation was observed,characterizing necrotic cell death(Fig.6g).DiscussionAs a cell model,both L929-A and L929-N cell lines have been previously used to study TNF a-induced necrosis[9, 23,28].In this study,we report that TNF a-induced cell death in these two L929cell lines is different,with several important different characteristics summarized in Table1. L929-A cells display features suggesting that TNF a alone induces cell death;however,the dead cells did not show sole AnnexinV positivity but were AnnexinV/propidium iodine(PI)-double positive,and their cell death was blocked by the RIP1kinase inhibitor Nec-1,a cell death that is easily miscategorized as necrosis[23].Compared with the L929-N cells,the L929-A cells differed in their response to the pan-caspase inhibitor,Z-VAD,and Q-VD did not induce but blocked TNF-induced cell death.Several studies have reported that the mechanism by which Z-VAD induces either autophagic cell death or necrosis in L929-N cells is via initiating autocrine TNF a[31,42],and the effect of autocrine TNF a has also been conﬁrmed in cad-mium exposure-triggered necrosis[43].These data indicate that the autocrine production of TNF a plays an important Fig.4Effect of TNF a-induced JNK activation,ROS accumulation, and calpain and cathepsin activity on Bid cleavage.a The effect of SP600125on TNF a-induced L929-A cell death and Bid cleavage. The cells were treated with TNF a in the absence or presence of 40l M SP600125for24h.The cells were stained with Annexin V-FITC and PI and analyzed byﬂow cytometry.The mitochondria and cytoplasm were separated,and the proteins were extracted. Western blotting was used to detect Bid cleavage.*P\0.01.b, c BHA and NAC differentially regulated TNF a-induced Bid cleavage and cell death in L929-A cells.The cells were treated with TNF a in the absence or presence of100l M BHA or5mM NAC for24h.Cell death was determined by staining the cells with PI and analyzed by ﬂow cytometry.Western blotting was used to detect Bid cleavage. *P\0.01.d The effect of E-64-c on TNF a-induced L929-A cell death and Bid cleavage.The cells were treated with TNF a in the absence or presence of E-64-c(16l g/mL)for24h.Cell death and Bid cleavage were detected as described above.e ALLN,pepstatin A and TPCK have no effect on TNF a-induced cell death in L929-A cells.The L929-A cells were treated TNF a in the absence or presence of ALLN(20l M),pepstatin A(10l M)and TPCK(20l M)for24h. Cell death was examined by microscopy(9200)and analyzed byﬂow cytometrycApoptosisrole in mediating necrosis,at least in L929-N cells.Unlike L929-N cells,which have high levels of constitutive and Z-VAD-inducible TNF a transcription,the L929-A cells only had a low level of TNF a expression that was barely enhanced by Z-VAD stimulation,the absence of autocrine TNF a may explain why Z-VAD failed to kill the L929-A cells.The key role of RIP3in mediating necroptosis has been shown in many studies;however,recent studies have also found that RIP3expression is important for caspase inhibitors to switch the apoptotic response to necrosis[14] and that RIP3protein kinase activity is essential for nec-roptosis but also governs whether a cell activates Caspase-8and dies by apoptosis[32].The L929-A cells express comparatively low levels of RIP3;therefore,thisprotein Apoptosis。

siRNA的转染

siRNA的转染将制备好的siRNA，siRNA表达载体或表达框架转导至真核细胞中的方法主要有以下几种:1。

磷酸钙共沉淀将氯化钙,RNA(或DNA)和磷酸缓冲液混合，沉淀形成包含DNA且极小的不溶的磷酸钙颗粒。

磷酸钙-DNA复合物粘附到细胞膜并通过胞饮进入目的细胞的细胞质。

沉淀物的大小和质量对于磷酸钙转染的成功至关重要。

在实验中使用的每种试剂都必须小心校准，保证质量,因为甚至偏离最优条件十分之一个pH都会导致磷酸钙转染的失败。

2.电穿孔法电穿孔通过将细胞暴露在短暂的高场强电脉冲中转导分子.将细胞悬浮液置于电场中会诱导沿细胞膜的电压差异，据认为这种电压差异会导致细胞膜暂时穿孔。

电脉冲和场强的优化对于成功的转染非常重要，因为过高的场强和过长的电脉冲时间会不可逆地伤害细胞膜而裂解细胞。

一般，成功的电穿孔过程都伴随高水平(50%或更高）的毒性。

3.DEAE-葡聚糖和polybrene带正电的DEAE-葡聚糖或polybrene多聚体复合物和带负电的DNA分子使得DNA 可以结合在细胞表面。

通过使用DMSO或甘油获得的渗透休克将DNA复合体导入。

两种试剂都已成功用于转染。

DEAE-葡聚糖仅限于瞬时转染.4。

机械法转染技术也包括使用机械的方法，比如显微注射和基因枪(biolistic particl e）。

显微注射使用一根细针头将DNA，RNA或蛋白直接转入细胞质或细胞核。

基因枪使用高压microprojectile将大分子导入细胞。

5。

阳离子脂质体试剂在优化条件下将阳离子脂质体试剂加入水中时，其可以形成微小的（平均大小约100-400nm）单层脂质体.这些脂质体带正电，可以靠静电作用结合到DNA的磷酸骨架上以及带负电的细胞膜表面。

因此使用阳离子脂质体转染的原理与以前利用中性脂质体转染的原理不同.使用阳离子脂质体试剂，DNA并没有预先包埋在脂质体中，而是带负电的DNA自动结合到带正电的脂质体上，形成DNA—阳离子脂质体复合物。

siRNA转染步骤—悬浮细胞

siRNA转染步骤—悬浮细胞
6孔板实验为例：
1、转染的当天，收集细胞离心，用含FBS的培养基重悬。

2、在250μl的无血清生长培养基中加入100 pmole siRNA，柔和混匀。

3、Lipofectamine RNAiMAX使用前要先混匀，然后在250 μl 生长培养基中加入5μl的Lipofectamine RNAiMAX进行稀释，室温下温育5分钟。

4、混合稀释好的siRNA和Lipofectamine RNAiMAX，室温下温育20分钟。

5、加入1.5 mL细胞悬浮液，每孔终体积为2ML，并轻晃混匀。

根据细胞数量决定于细胞类型和转染后分析测试的时间。

6、细胞在CO2培养箱中37℃温育24-48小时后，进行转染后的其它检测步骤。

如果细胞株比较敏感，孵育5-6 小时后，除去复合物，更换培养基。

sirna转染实验步骤及实验要点

sirna转染实验步骤及实验要点siRNA转染实验步骤如下：1.细胞接种：提前一天将细胞种植在24孔板中，以转染时细胞汇合度在30%左右为宜，转染前全培养基总量为0.45ml。

2.转染过程：•取0.67μg (50pmol) 的siRNA，加入一定量无血清稀释液，充分混匀，制成RNA稀释液，终体积为25μl。

注意：无血清稀释液建议采用OPTI-MEM、无血清DMEM或1640。

•取1μl的EntransterTM-R4000，然后加入24μl无血清稀释液体，充分混匀，制成EntransterTM-R4000稀释液，终体积为25μl。

室温静置5分钟。

•将EntransterTM-R4000稀释液和RNA稀释液充分混合（可用振荡器振荡或用加样器吹吸10次以上）混合，室温静置15分钟。

转染复合物制备完成。

•将50μl转染复合物滴加到有0.45ml全培养基（可含10%血清和抗生素）的细胞上，前后移动培养皿，混合均匀。

注意：对本试剂，采用含血清的全培养基有助于提升转染效率。

•转染后6小时观察细胞状态，如状态良好可不必更换培养基，继续培养24-96小时得到结果。

3.观察和检测：根据具体实验需求，可以在转染后的不同时间点观察细胞状态、检测基因表达、蛋白质表达等。

实验要点：1.细胞接种密度要适宜，一般在30%左右汇合度较好。

2.无血清稀释液的选择对于siRNA的稳定性和转染效率至关重要。

建议采用OPTI-MEM、无血清DMEM或1640等品牌。

3.在制备转染复合物时，要保证各个步骤的混合均匀，避免产生气泡。

4.在将转染复合物加入细胞时，要保证细胞的生存环境，避免对细胞造成损伤。

5.在转染后的观察和检测中，要注意保证实验结果的准确性和可靠性。

以上信息仅供参考，建议查阅专业文献获取更准确的信息。

siRNA细胞培养步骤

悬浮细胞的siRNA转染操作步骤悬浮细胞的siRNA转染操作步骤，以24孔板siRNA转染为例方法/步骤1. 11.提前1天细胞种植采用对数生长期的悬浮细胞，数量为常规培养细胞数的1/3进行转染实验。

如某细胞常规培养的细胞数是6×105，那么就用2×105的细胞进行转染。

END方法/步骤21. 12.转染过程⑴取0.67ug(50pmol)的siRNA，加入一定量无血清稀释液，充分混匀，制成RNA稀释液，终体积为25μl。

注意：无血清稀释液建议采用OPTI-MEM、无血清DMEM或1640。

2. 2⑵取1ul的EntransterTM-R4000，然后加入24ul无血清稀释液体，充分混匀，制成EntransterTM-R4000稀释液，终体积为25μl。

室温静置5分钟。

3. 3⑶将EntransterTM-R4000稀释液和RNA稀释液充分混合（可用振荡器振荡或用加样器吹吸10次以上）混合，室温静置15分钟。

转染复合物制备完成。

4. 4⑷将50μl转染复合物滴加到有0.45ml全培养基（可含10%血清和抗生素）的细胞上，前后移动培养皿，混合均匀。

5. 5⑸转染后6小时观察细胞状态，如状态良好可不必更换培养基，继续培养24-96小时得到结果。

END注意事项∙siRNA转染后，继续培养24-72小时在mRNA水平得到结果，继续培养24-96小时在蛋白水平得到结果。

mRNA转染后，根据需要在24小时后得到结果。

∙在部分实验室，由于血清和培养条件等差异，转染后镜下培养基中可能出现少量黑点状沉淀，为转染试剂和血清中蛋白结合产物，不影响转染结果和细胞状态，可通过换液除去。

siRNA转染方案

siRNA转染方案-向六孔板的每个孔种植2 x 10^5个细胞，每孔加入2ml不含抗生素的培养基注意：这个方案是针对六孔板中的一个孔，根据孔或皿的大小决定细胞和试剂的量。

-在含有CO2的37° C孵箱中培养细胞至60-80%融合。

这大概需要18—24小时。

注意：健康和不完全融合的细胞对于成功转染试验是必要的。

建议转染前一天要确保细胞存活。

-准备以下溶液：溶液A：每次转染，稀释2-8 μl siRNA duplex（例如0.25-1μg 或20-80pmol siRNA）至100 μl siRNA 转染基sc-36868溶液B：每次转染，稀释2-8 μl siRNA转染试剂sc-29528至100 μl siRNA转染基sc-36868。

6 μl siRNA 转染试剂可达高峰反应注意：不要向siRNA转染基sc-36868内添加血清和抗生素注意：最佳siRNA转染使用量对于不同目标蛋白是不同的，需通过实验来决定注意：如果需要低浓度siRNA，可用siRNA稀释缓冲液sc-29527稀释注意：尽管siRNA 转染试剂sc-29528对很多细胞系都是高效的，但可能不适用于所有细胞-直接将溶液A加入溶液B，然后用吸管上下轻柔吹打，室温下孵育15-45分钟-用2ml siRNA转染基sc-36868清洗细胞一次，吸出转染基立即进入下一步骤-每次转染，向溶液A和溶液B混合液中加入0.8ml转染基。

轻柔混合后用混合液覆盖冲洗过的细胞-将细胞放在含CO2的37℃孵箱孵育5-7小时注意：有些细胞可能需要长时转染。

然而长时血清饥饿可能会导致细胞分离或凋亡。

注意：结合了荧光素的对照siRNA只能在含CO2的37℃孵箱孵育5-7小时，孵育结束后利用荧光显微镜检测-加入1ml两倍血清浓度和抗生素浓度的正常培养基(2x正常培养基)，不要移除转染混合液。

如果有细胞毒性，移除转染混合液，用1x正常培养基替代-将这些细胞再孵育18-24小时-吸出培养基，用新鲜1x正常培养基取代-加入新鲜培养基24-72小时后利用合适的方案检测细胞注意：在siRNA试验中应该设立对照。

siRNA 使用说明

siRNA 使用说明siRNA 使用说明1.介绍siRNA（小干扰RNA）是一种能够特异性沉默靶基因表达的双链RNA分子。

本文档旨在提供关于siRNA的详细使用说明，包括实验前准备、实验步骤和数据分析等内容。

2.实验前准备2.1 选择siRNA：选择适合的siRNA靶向靶基因。

可以通过文献研究或生物信息学预测来确定siRNA的序列。

2.2 siRNA合成与纯化：合成和纯化siRNA应该由可靠的供应商进行。

确保合成的siRNA纯度高且无附加污染物。

2.3 细胞培养：选用适当的细胞系，并且按照常规细胞培养方法将其培养在适宜的培养基中。

3.实验步骤3.1 载体转染：将siRNA与特定的转染试剂混合，并按照转染试剂的说明书将其转染入细胞中。

注意控制组设置。

3.2 RNA干扰效率检测：根据靶基因的表达情况选择适当的方法检测siRNA的干扰效果，如定量PCR或Western blot等。

3.3 功能检测：根据实验需要，选择适当的功能检测方法，如细胞增殖、凋亡、迁移或侵袭等。

4.数据分析4.1 干扰效果分析：对实验结果进行统计学分析，比较siRNA处理组和对照组之间的差异，并计算干扰效果的百分比。

4.2 功能检测结果分析：对功能检测结果进行统计学分析，比较siRNA处理组和对照组之间的差异，并进一步解释其生物学意义。

5.附件本文档涉及的附件包括实验记录表、数据分析表和相应的图表。

请在需要时参考附加的文件。

6.法律名词及注释6.1 siRNA：小干扰RNA，一种双链RNA分子，用于特异性沉默靶基因表达。

6.2 RNA干扰：通过siRNA分子的特异性结合和降解，抑制靶基因的转录和翻译过程。

6.3 转染：将外源DNA或RNA导入靶细胞以实现外源基因的表达或干扰基因的沉默。

6.4 培养基：一种用于细胞培养的含有营养物质和生长因子的液体或固体培养基质。

7.结束语感谢您阅读本siRNA使用说明，希望本文档能帮助您顺利进行siRNA实验。

siRNA转染 Protocol

siRNA转染实验是一种常用的基因沉默技术，其基本原理是通过将小干扰RNA（siRNA）引入细胞，从而在翻译水平上抑制特定基因的表达。

以下是siRNA转染的实验原理、所需试剂和耗材、实验仪器、准备工作、实验方法、注意事项、常见问题及解决方法。

一、实验原理siRNA是一种21-23个核苷酸长的双链RNA，它与靶基因的mRNA 序列互补，通过碱基配对原则与mRNA结合，抑制基因表达。

siRNA 转染实验是通过将siRNA转入细胞内，利用细胞内自然存在的RNA 干扰机制，在转录后水平抑制基因表达。

这种技术具有高效性、特异性和可逆性等特点，被广泛应用于基因功能研究、药物筛选和疾病治疗等领域。

二、所需试剂和耗材1.试剂：o siRNA：针对特定基因的siRNA分子。

o转染试剂：如Lipofectamine、JetPrime等，用于将siRNA转入细胞。

o培养基：如DMEM、F12等，用于细胞培养。

o血清：如胎牛血清，提供细胞生长所需的营养物质。

o抗生素：如青霉素、链霉素等，用于防止细胞污染。

2.耗材：o细胞培养瓶、板：用于细胞培养。

o离心管：用于离心和分离细胞。

o移液器及枪头：用于精确加样。

o过滤器：用于过滤溶液中的杂质。

o无菌水：用于稀释和配制溶液。

三、实验仪器1.实验室搅拌器：用于混合溶液。

2.高速冷冻离心机：用于离心和分离细胞。

3.水浴锅：用于加热溶液。

4.无菌工作台或超净工作台：用于进行无菌操作。

5.分光光度计：用于测量细胞生长状况和转染效率。

6.荧光显微镜：用于观察细胞转染后荧光蛋白的表达情况。

7.CO2培养箱：提供细胞培养所需的气体环境。

8.显微镜：观察细胞的生长状态和siRNA转染后的细胞变化。

9.细胞计数板或细胞计数仪：用于细胞计数，确定细胞的密度和生长状态。

10.酶标仪或多功能读板仪：用于检测细胞因子的浓度。

四、实验准备工作1.确认细胞系和siRNA：实验前要明确所使用的细胞系以及针对的目标基因。

siRNA转染实验指导

siRNA转染RNA干扰现象是由与靶基因序列同源的双链RNA引发的，广泛存在于生物体内的序列特异性基因转录后的沉默过程。

胞质中的核酸内切酶Dicer将dsRNA裂解成由21-25个核苷酸组成的siRNA，随后siRNA与体内蛋白结合形成RNA诱导的沉默复合物RISC，RISC与外源性基因表达的mRNA的同源区进行特异性结合，在结合部位切割mRNA，被切割后的断裂mRNA 随即降解，从而阻断相应基因表达的转录后基因沉默机制。

用RNAi特异性地抑制如艾滋病病毒基因、肝炎病毒基因、癌基因等相关基因的过度表达，使这类基因保持在静默或休眠状态，这种技术已经成为研究基因功能的重要工具，并将在病毒病、遗传性疾病和肿瘤病的治疗方面发挥重要作用。

本次实验采用罗氏的X-tremeGENE siRNA转染试剂进行实验。

一、细胞准备转染前一天，取出处于对数生长期的细胞，吸掉原来的培养液，用无菌PBS清洗细胞。

加入1ml胰酶消化液消化细胞，显微镜下观察所有细胞完全皱缩变圆后加入终止液终止消化。

收集细胞悬液于15ml离心管中，800r/min室温离心5min。

用罗氏CASY快速细胞计数及活率分析仪进行细胞计数。

弃去上清，加入无抗生素培养基重悬细胞。

以1~4×105细胞/孔的密度接种到6孔培养板上，无抗生素培养基培养一天。

细胞转染前需用高质量血清进行培养，使细胞健康，否则将造成细胞营养不良，细胞呈现瘦削拉长状态，也会影响转染效率。

转染前3小时，取出无抗生素培养的细胞置于倒置显微镜下观察。

若融合率达50％～70％，即可进行转染实验。

二、耗材及试剂准备实验开始前将枪头、去核酸酶的EP管、试管架等放入无菌操作台，以紫外线照射30min。

转染前温育Optimen I Reduced Serum Medium，以及将X-tremeGENE siRNA转染试剂、siRNA 置于15-25度室温条件下平衡。

取出转染试剂，对EP管做好标记。

RNA转染试剂,高效率转染

Entranster TM-R4000（用于将RNA转染入动物细胞）使用手册一产品介绍本品推荐用于将siRNA、microRNA(miRNA)、mimic、inhibitor等小片断RNA转染入动物细胞（包括各种细胞系、原代细胞、悬浮细胞、昆虫细胞等）。

本品在多种细胞系的验证中均表现了很好的RNA转染效率，并有很低的细胞毒性，而较低的细胞毒性对RNAi实验尤其重要。

二重要提示1.由于本品细胞毒性很低，所以采用本品进行转染的细胞数量相对较少，这有助于提高转染效率。

2.采用本品进行转染，RNA用质量(ug)进行计量，对21nt双链的siRNA来说，1OD=3.0nmol=40ug。

3.siRNA等较短RNA请参照表1中siRNA用量，mRNA、shRNA等较长RNA请参照表1中mRNA用量。

4.如转染后需要用Trizol提取RNA，建议转染后6小时换液。

三实验过程（以24孔板siRNA转染为例）1.提前1天细胞种植贴壁细胞：提前一天将细胞种植在24孔板中，以转染时细胞汇合度（Confluence）在30％左右为宜，转染前全培养基总量为0.45ml。

悬浮细胞：采用对数生长期的细胞，数量为常规培养细胞数的1/3进行转染实验。

如某细胞常规培养的细胞数是6×105，那么就用2×105的细胞进行转染。

2.转染过程⑴取0.67ug(50pmol)的siRNA，加入一定量无血清稀释液，充分混匀，制成RNA稀释液，终体积为25μl。

注意：无血清稀释液建议采用OPTI-MEM、无血清DMEM或1640。

⑵取1ul的Entranster TM-R4000，然后加入24ul无血清稀释液体，充分混匀，制成Entranster TM-R4000稀释液，终体积为25μl。

室温静置5分钟。

⑶将Entranster TM-R4000稀释液和RNA稀释液充分混合（可用振荡器振荡或用加样器吹吸10次以上）混合，室温静置15分钟。

siRNA转染步骤—贴壁细胞

siRNA转染步骤—贴壁细胞
示例：使用Lipofectamine RNAiMAX和HeLa cell 用于siRNA 的在6孔板上进行的转染实验。

注：请实验前确认实验使用的细胞系是否适合转染试剂。

1、转染前一天，在6孔板中每个孔内播种 3.0 x 10^5 HeLa cells，每孔放2.5mL不含抗生素的生长培养基。

这样在转染时会有50-60%的融合率。

2、从细胞中去除生长培养基。

3、加入1.5 mL新鲜的不含血清的生长培养基。

4、对于每个转染的孔，按照如下方法准备siRNA 和Lipofectamine RNAiMAX 混合物。

4-1、在250μl的无血清生长培养基中加入100pmole siRNA，柔和混匀。

4-2、Lipofectamine RNAiMAX使用前要先混匀，然后在250μl 生长培养基中加入5μl的Lipofectamine RNAiMAX进行稀释，室温下温育5分钟。

4-3、混合稀释好的siRNA和Lipofectamine RNAiMAX，室温下温育20分钟。

5、加入混合物到含有HeLa cells的6孔板中，每孔终体积为2ML，并轻晃混匀。

6、在37℃的二氧化碳培养箱中培养细胞 5-6小时。

7、更换含有血清的培养基并培养细胞24-48小时，直到进行基因敲除实验。

细胞转染(Entranster)注意事项

细胞转染注意事项
1.如为贴壁生长细胞，一般要求在转染前一日，必须应用胰酶处理成单细胞悬液，重新接种于培养皿或瓶，转染当日的细胞密度以70-90%（贴壁细胞）或
2×106-4×106细胞/ml（悬浮细胞）为宜，最好在转染前4h换一次新鲜培养液。

2.用于转染的质粒DNA必须无蛋白质，无RNA和其他化学物质的污染，OD260/280比值应在1.8以上。

3.培养基中的血清
在开始准备DNA和阳离子脂质体试剂稀释液时要使用无血清的培养基，因为血清会影响复合物的形成。

其实，只要在DNA-阳离子脂质体复合物形成时不含血清，在转染过程中是可以使用血清的。

4.培养基中的抗生素
抗生素是影响转染的培养基添加物。

这些抗生素一般对于真核细胞无毒，但阳离子脂质体试剂增加了细胞的通透性，使抗生素可以进入细胞。

这降低了细胞的活性，导致转染效率降低。

这时候可以选择英格恩生物的Entranster转染试剂，非脂质体试剂，培养基可加抗生素，避免了细胞染菌。

5.设置阳性对照和阴性对照。

6.一般在转染24-48h，靶基因即在细胞内表达。

根据不同的实验目的，24-48h 后即可进行靶基因表达的检测实验。

7.如若建立稳定的细胞系，则可对靶细胞进行筛选，根据不同基因载体中所含有的抗性标志选用相应的药物，常用的真核表达基因载体的标志物有潮霉素和新霉素等。

原代细胞siRNA转染流程

原代细胞siRNA转染流程
Product Description RFectPM siRNA transfection reagent is a new generation of animal-origin free lipid transfection reagent specially designed for transfecting RNAi duplexes (siRNA) into eukaryotic cells. RFectPM has been formulated for high performance in multiple cell lines and primary cells. SiRNA-RFectPM complexes can be added directly to cells in culture medium, in the presence or absence of serum. It is not necessary to remove complexes or change/add medium after transfection but complexes may be removed after 4-6 hours. Advantages of RFectPM siRNA Transfection Reagent ￭ High transfection efficiency for primary cells with the rate of 80% or more and obvious effect of gene knockdown(eg.The efficiency of Lamin A/C gene knockout in liver cells was more than 90% ). ￭ Minimal cytot

DNA转染试剂-Entranster

Entranster TM-H4000（用于DNA的细胞转染）使用手册本品推荐用于常规细胞的转染。

一重要提示1.本品在转染过程中可以添加抗生素，抗生素的添加与否不影响转染效率和毒性。

2.如转染后需要用Trizol提取RNA，建议转染后6小时换液。

二实验过程（以24孔板为例）1.提前一天细胞铺板提前一天将细胞种植在24孔板中，以转染时细胞密度在60％左右为宜。

2.转染过程⑴将0.8μg的DNA用25μl无血清稀释液稀释，充分混匀，制成DNA稀释液。

注意：无血清稀释液建议采用OPTI-MEM、无血清DMEM或1640。

⑵将2μl的Entranster TM-H4000用25μl无血清稀释液体稀释，充分混匀，制成Entranster TM-H4000稀释液。

室温静置5分钟。

⑶将Entranster TM-H4000稀释液分别加入到DNA稀释液中，充分混匀（可用振荡器振荡或用加样器吹吸10次以上），室温静置15分钟。

转染复合物制备完成。

⑷将转染复合物加入到含细胞和完全培养基的培养容器上，轻柔混匀。

注意：①对本试剂，采用含血清的全培养基有助于提升转染效率。

②完全培养基可加抗生素。

⑸培养4-6小时后更换培养基，继续培养24-48小时。

注意：①如细胞没有毒性等不良情况，转染后4-6小时可不必更换培养基。

注意：如用于同时共转染多种质粒，DNA的用量指每种质粒用量的总和，这时如需得到较高转染效率，建议将DNA的用量和转染试剂的用量同步提高1.5-2倍。

三优化由于DNA和转染试剂的用量比值是决定转染效率的重要因素，同时由于各实验室质粒的定量误差，质粒纯化程度不同以及细胞状态不同，造成不同细胞和实验室的最优实验条件的差异，为取得最高的转染效率，初次应用时，建议先进行优化。

最优条件确定后，实验的结果将非常稳定。

下表列出了在6-well的优化方案，供参考。

根据预实验的最优化条件，按培养器皿表面积比例应用到其他培养容器。

四常见问题与解决方案。

荧光标记的siRNA转染方法及步骤

荧光标记的siRNA转染方法及步骤一、荧光标记的siRNA转染效率的高低可以通过观察荧光标记(FAM、CY3、CY5)的siRNA转染细胞后的荧光强度判断。

荧光标记的siRNA转染细胞后，可以用荧光显微镜、激光共聚焦显微镜、流式细胞仪等检测，确定转染是否有效及转染条件。

荧光标记的siRNA还可用作siRNA胞内定位及双标记实验(配合标记杭体)来追踪转染过程中导入了siRNA的细胞，将转染与靶蛋白表达的下调结合起来。

二、使用荧光标记的siRNA检测转染效率荧光标记的siRNA的溶解及保存1.由于OligoRNA是很轻的干膜状附在管壁上，打开时极易散失，所以打开离心管前先离心，10000 rpm，2 min然后再慢慢打开管盖，溶解时加适量DEPC 水后盖上管盖，振荡溶解。

2.需要浓度20μM的样品，如何计算重悬siRNA缓冲液的量？1 OD的siRNA，加入125μl附送的DEPC水,溶解后终浓度为20μM。

3.贮存和稳定性：-20°C ,避光保存，冻干粉或液体。

液体(贮存浓度为20μM)避免反复冻融，GenePharma保证在上述条件下siRNA oligo冻干粉的稳定性可达到6个月，液体状态下建议3个月内使用完。

转染1.使用lipo fectamin2000转染的步骤(以贴壁细胞为例，仅供参考)a.以24孔培养板操作为例(其他孔板各种的试剂用量，请参照“Lipofectamine2000manual”)，转染前一天，将0.5-2×105个细胞接种于培养板中，每孔中加入约500μl含血清的完全培养基，使转染时的细胞密度能够达到70%；b.取1μl/孔Lipo fectamine2000(使用前轻轻摇匀)，用50μl Opti-MEM I ReducedSerum Medium稀释。

轻轻混和后在室温孵育5 min ;c.取2μl荧光-siRNA，用50μl Opti-MEM I Reduced Serum Medium稀释，轻轻混和均匀;d.稀释的Lipofectamine2000(步骤b)经过5min的孵育后，与稀释荧光-siRNA(步骤c)轻轻混和，室温静置20min，以形成荧光-siRNA-转染试剂混和物，如果溶液出现浑浊，属于正常现象，不会影响转染效果。

RNA转染研究病理性色素沉着(entranster),转染效率高

A novel P53/POMC/G a s/SASH1autoregulatory feedback loopactivates mutated SASH1to cause pathologichyperpigmentationDing’an Zhou a,b *,Zhiyun Wei c ,Zhongshu Kuang a ,Huangchao Luo a ,Jiangshu Ma a ,Xing Zeng a ,Ke Wang a ,Beizhong Liu a ,Fang Gong a ,Jing Wang a ,Shanchuan Lei a ,Dongsheng Wang d ,Jiawei Zeng e ,Teng Wang b ,Yong He a ,Yongqiang Yuan a ,Hongying Dai a ,Lin He b,c ,Qinghe Xing b,*aDepartment of Laboratory Medicine,Yongchuan Hospital,Chongqing Medical University,Chongqing,ChinabChildren’s Hospital and Institutes of Biomedical Sciences,Fudan University,Shanghai,ChinacBio-X Institute,Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders (Ministry of Education),Shanghai Jiao Tong University,Shanghai,ChinadDepartment of Laboratory Medicine,The Afﬁliated Hospital of North Sichuan Medical College,Nanchong,ChinaeDujiangyan People’s Hospital,Cheng du,Sichuan,ChinaReceived:August 4,2016;Accepted:September 28,2016Abstractp53-Transcriptional-regulated proteins interact with a large number of other signal transduction pathways in the cell,and a number of positive and negative autoregulatory feedback loops act upon the p53response.P53directly controls the POMC/a -MSH productions induced by ultra-violet (UV)and is associated with UV-independent pathological pigmentation.When identifying the causative gene of dyschromatosis univer-salis hereditaria (DUH),we found three mutations encoding amino acid substitutions in the gene SAM and SH3domain containing 1(SASH1),and SASH1was associated with guanine nucleotide-binding protein subunit-alpha isoforms short (G a s).However,the pathological gene and pathological mechanism of DUH remain unknown for about 90years.We demonstrate that SASH1is physiologically induced by p53upon UV stimulation and SASH and p53is reciprocally induced at physiological and pathophysiological conditions.SASH1is regulated by a novel p53/POMC/a -MSH/G a s/SASH1cascade to mediate melanogenesis.A novel p53/POMC/G a s/SASH1autoregulatory positive feedback loop is regu-lated by SASH1mutations to induce pathological hyperpigmentation phenotype.Our study demonstrates that a novel p53/POMC/G a s/SASH1autoregulatory positive feedback loop is regulated by SASH1mutations to induce pathological hyperpigmentation phenotype.Keywords:SASH1 p53 DUH hyperpigmentationIntroductionThe skin pigmentation is originated from the synthesis of melanin in the melanocytes,followed by distribution and transport of pig-ment granules to neighbouring keratinocytes [1].Variations in the coding region of the melanocortin-1-receptor (MC1R)are impor-tant for tanning and pigmentation in human beings.MC1R is a G protein-coupled receptor (GPCR)that is preferentially expressed in epidermal melanocytes [2]and is activated by its ligand a -melano-cyte-stimulating hormone (a -MSH),a propigmentation hormone which is produced and secreted by both keratinocytes andmelanocytes in the skin following UV.a -melanocyte-stimulating hormone and other bioactive peptides are cleavage products of pro-opiomelanocortin (POMC),a multi-component precursor for a -MSH (melanotropic),ACTH (adrenocorticotropic)and the opioid peptide b -endorphin.Normal synthesis of a -MSH and ACTH is an important determinant of constitutive human pigmentation and the cutaneous response to UV [2].Within melanocytes,MC1R regulates the amount and type of pig-ment production and is a major determinant of skin phototype,*Correspondence to:Prof.Qinghe XING E-mail:qhxing@ Dr.Ding’an ZHOUE-mail:081023094@ª2016The Authors.Journal of Cellular and Molecular Medicine published by John Wiley &Sons Ltd and Foundation for Cellular and Molecular Medicine.This is an open access article under the terms of the Creative Commons Attribution License,which permits use,distribution and reproduction in any medium,provided the original work is properly cited.doi:10.1111/jcmm.13022J.Cell.Mol.Med.Vol 21,No 4,2017pp.802-815sensitivity to UV radiation-induced damage and skin cancer risk[3]. Upon ligand binding,GPCRs impart a signal to heterotrimeric G proteins, which are composed of a-,b-and c-subunits,resulting in the detach-ment of the a-subunit from the G bc subunit of G proteins.G proteins of the G a s class directly catalyse the transformation of ATP to cAMP. cAMP is responsible for melanogenic actions of such ligands as a-MSH,including the activation of tyrosinase in melanin biosynthesis[4].The tumour-suppressor protein p53,a transcriptional factor,has been documented to directly activate transcription of numerous genes such as those that control cell-cycle,apoptosis and others.P53 directly mediates UV induction of POMC/MSH in skin and stimulates the POMC promoter in response to UV and is involved in UV-indepen-dent pathologic pigmentation and could mimic the tanning response [1].In the skin,p53function is critical for the retention of tissue integrity following UV irradiation[1].UV can exclusively induce dipyrimidine C to T substitutions that include CC to TT frameshift mutations in the p53gene,which were found in the skin of UV-irra-diated mice months before tumour development[5].In addition to the above activities,p53has been shown to be essential for the for-mation of‘sunburn cells’,which are a hallmark of sunburns[5].DUH is a clinically heterogeneous disorder that is characterized by generalized mottled pigmentation.DUH was initially described by Ichi-kawa and Hiraga in two generations of two families in1933[6].We discovered similar Chinese DUH pedigrees with dyschromatosis sym-metrica hereditaria(DSH)in2003with autosomal-dominant DUH[7] and diagnosed as DUH rather than DSH subsequently.However,the pathological gene and pathological mechanism of DUH have not been further characterized since itsﬁrst report in1933.SASH1was originally described as a candidate tumour-suppressor gene in the carcinomas of breast and colon and belongs to the previ-ously described novel family of putative adapter and scaffold proteins that transfer signals from the ligand to the receptor[8–10].Our previ-ousﬁndings indicate that SASH1binds to G a s,the downstream mole-cule of a-MSH/MC1R signalling cascade[11].Our previous study also showed that,in several DUH several affected individuals,hyperpig-mented macules were showed to become more pronounced after strong UV exposure especially in summer[7],but no further mechanism was identiﬁed the reasons of photosensitivity[12].The importance of expression of p53/POMC/a-MSH in UV-photopigmentation response and UV-independent hyperpigmentation has been elucidated[1].More-over,less observations were reported to demonstrate that the variations in SASH1gene are associated with hyperpigmentation and how these variations cause hyperpigmentation.Taken above,we hypothesise that a novel p53/POMC/a-MSH/G a s that SASH1is involved in,to mediate UV-photopigmentation response and pathological hyperpigmentation.Materials and methodsPCR,sequencing and mutation analysisTwo Chinese families from the Henan and Yunnan provinces of China and one American family with typical features of DUH were recruited for this study.Three pedigrees with DUH showed an autosomal-domi-nant inheritance pattern and were ascertained by experienced clinical dermatologists.The American family is a smaller pedigree,which could only provide three peripheral blood samples from affected indi-viduals for study.This research was approved by the ethical review committees from the appropriate institutions.Genotyping was per-formed,and the two-point LOD score was calculated as previously described[7].In total,50family members and500normal individuals (controls)participated in the study after providing informed consent. Samples of peripheral blood DNA were taken from all available family members.PCR and sequencing were performed as previously described [7].The sequencing was performed with an ABI BigDye Terminator Cycle Sequencing Kit(Applied Biosystems Inc,Foster City,CA,USA)on an ABI PRISM3130DNA Analyzer(Applied Biosystems),and data were analysed using sequence analysis software,version3.4.1(Applied Biosystems). Sequence data were compared with the SASH1reference sequence(Gen-Bank NM_015278.30)using Sequencher4.10.1(Gene Codes Corp,Ann Arbor,Michigan,USA).Nucleotide numbering reﬂects complementary DNA (cDNA)numbering,with+1corresponding to the A of the ATG translation initiation codon in the reference sequence[7].Construction of SASH1,G a s,POMC and p53 expression vectorsThe construction protocol of recombined vector of wt and mutant SASH1-PEGFP-C3and wt and mutant SASH1-PBABE-Flag-puro was mainly referred to our previous study[11].To construct HA-Pcna3.0-p53,myc-Pcdna3.0-POMC and GFP-G a s-Pegfp-C3vectors,PCRs of bacteria (obtained from Han jiahuai Lab,Xiamen University,Xiamen,China)contain-ing the vector of full-length CDS sequences of G a s,p53and POMC were performed with Phusion Hot Start High Fidelity Polymerase(New England Biolabs,Inc.,Ipswich,Massachusetts,USA)or GXL Polymerase(Takara, Shimogyo-ku,Kyoto,Japan),and the following primers were used:G a s primers50-ACGCGTCGACATGGGCTGCCTCGGGAAC-30(forward,Sal I site included)and50-CCGCTCGAG TTAGAGCAGCTCGTACTGACG-30(reverse, Xho I site included);p53primers50-CGCGGATCCGCCACCACCATGGAGG AGCCGCAGTCAGATCCTA-30(forward,BamH I site included)and50-CCG CTCGAGTCAGTCTGAGTCAGGCCCTTCTGT(reverse,Xho I site included); POMC primers50-CGCGGATCC ATGCCGAGATCGTGCTGC-30(forward, BamH I site included)and50-CCCAAGCTTT CACTCGCCCTTCTTGTA GGCGTTCTTGAT-30(reverse,Xho I site included).Mammalian expression vectors(Invitrogen,Carlsbad,California,USA)via the relative restriction sites were sequenced.Cell culture and transfectionA375cells,SK-MEL-28cells and HEK-293T cells were maintained as previously described[13].Normal human epithelial melanocytes (NHEMs,C-12402;PromoCell,Germany)were cultured in M2medium. A375,SK-MEL-28and HEK-293T cells were transfected using Lipofec-tamine2000(11668-027;Invitrogen)as previously described[13,14] or Entranster-D(18668-01;Engreen Biosystem Co.,Ltd,New Zealand) or polyethyleneimine(PEI)prepared by ourselves.The transfected A375 and SK-MEL-28cells were cultured in1.5l g/ml puromycin or2.0l g/ ml G418to select stable cell lines.HEK-293T cells were transiently transfected with wild-type and mutant SASH1-pEGFP-C3or co-trans-fected with wild-type SASH1-Pbabe-Flag-puro and G a s-Pegfp-C3ª2016The Authors.Journal of Cellular and Molecular Medicine published by John Wiley&Sons Ltd and Foundation for Cellular and Molecular Medicine.803J.Cell.Mol.Med.Vol21,No4,2017vectors for immunoprecipitation experiments.NHEMs and HEK-293or HEK-293T cells were transiently transfected with Pcdna3.0-HA-p53, Pcdna3.0-myc-POMC,Pegfp-C3-G a s and wild-type SASH1-pEGFP-C3 according to pairwise combination to analyse the expression of exoge-nous p53,POMC,G a s and SASH1using PEI prepared by ourselves or PromoFectin(PK-CT-2000-MAC-1;PromoCell,Heidelberg,Germany).HEK-293T cells were transfected with G a s-GFP,HA-p53,myc-POMC and GFP-SASH1vector and subsequently silenced by G a s-and POMC-speciﬁc siRNAs that were synthesized by Shanghai GenePharma Co., Ltd(Shanghai,China)using Entranster TM-R Transfection Reagent (18668-06;Engreen Biosystem Co.,Ltd).The sense and antisense strands of each siRNA for G a s,POMC,GAPDH and the negative control are shown in Table S3.Pull-down assay and nano-ﬂow LC-MS/MS and bioinformatic analysisThe protocols for the pull-down assay,nano-ﬂow LC-MS/MS,database search and bioinformatic analysis for functional classiﬁcation are mainly referred to our previous report[11]. Immunoprecipitation and immunoblottingHEK-293T or HEK-293transfected cells and NHEMs were gently washed in PBS three times and then lysed ing IP-Western blot lysis buffer(P0013;Beyond Time BioScience and Tech-nology company,Jiangshu,China)in the presence of a complete protease inhibitor cocktail,1l M sodium orthovanadate and1mM sodiumﬂuoride per10-cm dish on ice.Cell lysates were transferred into 1.5-ml microcentrifuge tubes.Extracts were centrifuged for 10min.at13400g.at4°C.Then,600l l of supernatants was pre-cleaned with20l l of Protein A/G PLUS-Agarose(sc-2003;Santa Cruz Biotechnology,Inc,California,USA)for1hr,immunoprecipitated using6l l of GFP-Tag(7G9)mouse mAb(M20004,Shanghai Abmart,Inc.,Shanghai,China)or6l l of DYKDDDDK-Flag-Tag mouse mAb(M20008;Shanghai Abmart,Inc.)or6l l of HA-Tag mouse mAb(SG4110-25;Shanghai Genomics,Shanghai,China)at4°C for 10hr and mixed with20l l of Protein A/G PLUS-Agarose(sc-2003, Santa Cruz Biotechnology,Inc.)at4°C for4hr and assayed using co-immunoprecipitation or immunoprecipitation.The immunoprecipi-tates were washed with PBS three times and subjected to SDS-PAGE and Western blotting.The primary antibodies used in the Western blot analysis were GFP-Tag mouse Ab(M20004,Shanghai Abmart,Inc.), Flag-tag mouse mAb(M20008;Shanghai Genomics),anti-G a s rabbit polyclonal Ab(G7X105877;Gene Tex,Inc.,Irvine,CA,USA),myc-tag mAb(SG411-30,Shanghai Genomics)and HA-tag mouse mAb(SG4110-25,Shanghai Genomics),SASH1Rabbit mAb(A302-265A-1,Bethyl Laboratories,Inc.,Montgomery,Texas,USA),DYKDDDDK-Flag-Tag mouse mAb(M20008;Shanghai Abmart,Inc.),TYRP1(TA99)mouse mAb(Ab3312;Abcam,Cambridge,UK),Rab27a mouse mAb (H0005873-M01;Abnova,Taipei City,Taiwan),melanoma gp100Rabbit mAb(ab137062;Abcam,Cambridge,UK),GAPDH mouse mAb(M20005; Shanghai Abmart,Inc.)and anti-b-tubulin mouse mAb(M20005M; Shanghai Abmart,Inc.).Immunoblotting was performed as previously described[15].Immunohistochemical and immunoﬂuorescence staining,and melanin staining Immunohistochemical stainingWritten informed consent regarding tissue and data use for scientiﬁc purposes was obtained from all participating patients.Epithelial tissues from affected individuals with the Y551D SASH1mutation from pedi-gree family I wereﬁxed in10%formalin at4°C for24hr and then embedded in parafﬁn.Parafﬁn sections(5l m)were incubated at56°C overnight and then deparafﬁnized and rehydrated using xylene and an ethanol gradient.The sections were incubated with the SASH1mono-clonal antibody(A302-265A-1;Bethyl Laboratories,Inc.),Rabbit Anti-ACTH(7-23)antibody(bs-004R;biosynthesis biotechnology Co.,Ltd, Beijing,China),Mitf polyclonal antibody(BS1550;Bioworld Technology, Inc,Louis Park,MN,USA),HMB45monoclonal antibody(sc59305; Santa Cruz Biotechnology,Inc.),TYRP1(TA99)mouse mAb(Ab3312; Abcam),Rab27a mouse mAb(H0005873-M01;Abnova)and p53mon-oclonal antibody(kit-0010-2;biosynthesis biotechnology Co.,Ltd)as well as horseradish peroxidase-linked anti-rabbit and antimouse univer-sal secondary antibodies or FITC.Finally,sections were counterstained with haematoxylin and photographed under the positive position micro-scope BX51.Immunoﬂuorescence(IF)and confocal microscopyWild-type or mutant SASH1-A375stable cells were plated in6-well chamber slides and incubated at37°C for at least48hr.Indirect immunoﬂuorescence analysis was performed on A375cells expressing wild-type and mutant SASH1(s)in6-well chamber slides to assess SASH1localization.IF was performed as described previously using the following antibodies:SASH1Rabbit mAb(A302-265A-1;Bethyl Labora-tories,Inc.)and DYKDDDDK-Flag mouse mAb(M20008;Shanghai Genomics)[11].Melanin stainingParafﬁn sections(5l m)from epithelial tissues were incubated in an 80°C baking oven for30min and then kept at room temperature for 15min.Melanin staining was performed according to the manufac-turer’s protocol(GMS80023.3;GENMED SCIENTIFICS INC.,Shanghai, China)and observed under a light microscope.Quantitative real-time RT-PCRThe total RNA from the different groups of SK-MEL-28cells was iso-lated using TRIzol Reagent(Invitrogen).Reverse transcription was car-ried out according to the manufacturer’s protocol for the PrimeScript TM RT Reagent Kit(DRR037A;Takara)or PrimeScript RT reagent using the gDNA Eraser Kit(DRR047A;Takara)for qRT-PCR.The sense and anti-sense primer sequences for SASH1,TYRP1,Pmel17,Rab27a,G a s, POMC and GAPDH are presented in Table S3.The PCR products were conﬁrmed by agarose gel electrophoresis.Real-time PCR was per-formed using the Applied Biosystems7500System with SYBR Premix Ex Taq TM(DRR041A;Takara).The quantity of each mRNA was normal-ized to that of GAPDH mRNA.804ª2016The Authors.Journal of Cellular and Molecular Medicine published by John Wiley&Sons Ltd and Foundation for Cellular and Molecular Medicine.UV exposureThe human foreskin tissues from a14-year-old boy were exposed for enough time under an UV phototherapy instrument(NBUVB SS-05; Sigma-Aldrich,St.Louis,Missouri,USA)to reach the expected UV intensity,thenﬁxed in10%formalin and embedded in parafﬁn for immunohistochemistry analyses.We conformed to the guidelines of the World Medical Assembly(Declaration of Helsinki)to acquire the human foreskin tissues.In the case of in vitro UV experiments which mainly referred to the protocol of our institute[16],HEK-293T cells and NHEMs transiently transfected with myc-POMC were cultured to approximately70–80% conﬂuence in6-cm-diameter dishes and were irradiated with100mJ/ cm2UVC delivered via a HL-2000HybriLinker with a254-nm wave-length(Upvon)and followed by the indicated recovery time.Finally, cells were harvested to detect proteins’levels using immunoblot. Electrophoretic mobility shift assayThree probes binding with/without biotin,which targeted SASH1pro-moter,were synthesized.The sequence of probes was as follows:probe 1#50-GCCCAAGCTT TCACACTTGTTT-30,probe2#50-CCAAGACTTGCTA-GAAGGAACGAGTCG-30,probe3#50-CGTGGCCACCTAGACCCGAGGTG-30. Electrophoretic mobility shift assay was performed as described as the protocol provided with LightShiftâChemiluminescent EMSA Kit(20148; Thermo Scientiﬁc,Pierce Biotechnology,Rockford,USA). Statistical analysisThe data are presented as meanÆstandard error of the mean (S.E.M.)s.These data wereﬁrst analysed using the homogeneity of variance test and followed by the change of variable test.Statistical signiﬁcance was determined by a one-factor analysis of variance (ANOVA)with LSD correction on SPSS version16.0(IBM(International Business Machine))to generate the required P-values.Cartograms were plotted using GRAPHPAD PRISM5(GraphPad Software, Jolla,CA,USA)5.ResultsMutations in SASH1in DUH-affected individuals result in the up-regulation of SASH1in vitro and in vivoWe have located the gene that is responsible for DUH is local-ized to chromosome6q24.2-q25.2[7].The10.2-Mb region on chromosome6(6q24.2-q25.2)that isﬂanked by the markers D6S1703and D6S1708contained more than50candidate genes. We screened selected genes in this region for possible pathologi-cal mutations by directly sequencing the PCR products of exons that were ampliﬁed from genomic DNA of affected,unaffected and control individuals.We sequenced50candidate genes and found three heterozygous mutations encoding amino acid substi-tutions in SAM and SH3domain containing I(SASH1)in the probands in each of the two non-consanguineous Chinese DUH-affected families(families I and II)and in one non-consangui-neous American DUH-affected family(family III).SASH1point mutations were found in the three pedigrees.These mutations were as follows:a T?G substitution at nucleotide2126in exon 14in family I,a T?C substitution at nucleotide2019in exon 13in family II and a G?A substitution at position2000in exon 13in family III.These three nucleotide changes cause non-con-servative missense mutations in the SASH1gene,resulting in the following amino acid substitutions:Tyr to Asp at codon551 (TAC?GAC),designated as Y551D;Leu to Pro at codon515 (CTC?CCC),designated as L515P;and Glu to Lys at codon509 (GAA?AAA),designated as E509K(Fig.1A).These sequence changes were conﬁrmed in all of the affected family members but were not observed in unaffected family members,correlating the presence of the mutations with the presence of the pheno-type.The mutations were not observed in any of the500normal controls or in any of the current databases,including the Hap-Map database.Therefore,these mutations are unlikely to be common single nucleotide polymorphisms(SNPs)[7].When SASH1mutants were stably expressed in A375cells,they signiﬁcantly up-regulated SASH1(Fig.1B).Immunoblotting demon-strated that SASH1was up-regulated in A375cells stably express-ing either wild-type(WT-A375cells)or mutant SASH1(mutant-A375cells,including E509K-A375cells,L515P-A375cells and Y551D-A375cells),compared to the expression of endogenous SASH1in A375cells expressing the pBABE-puro empty vector (VECTOR-A375cells)or A375cells without any transfected vector (BLANK-A375cells)(Fig.1B).To verify the stability of SASH1pro-teins,HEK-293T cells stably expressing wild-type or mutant SASH1 were treated with20l g/ml of the protein synthesis inhibitor cyclo-heximide(CHX)for the indicated times to assess the half-life of SASH1.The protein levels of SASH1decreased in a time-course-dependent manner in response to CHX treatment.Wild-type SASH1 levels decreased with a half-life of approximately4hr.However,with CHX treatment for6hr or longer,CHX began to degrade mutant SASH1proteins.Therefore,the three mutant SASH1proteins were more stable than the wild-type,supporting the above observation that SASH1mutants are expressed at higher levels than the wild-type (Fig.S1A and B).Endogenous SASH1was an unstable protein with a half-life of approximately3hr(Fig.S1C).We characterized the subcellular localization of SASH1in A375cells and skin epithelial layers.The endogenous SASH1 protein in VECTOR-A375cells and the skin epithelial layers from normal controls demonstrated a homogeneous pattern of expres-sion(Fig.1C and Fig.S2-a4).However,in WT-A375cells and mutant-A375cells,activated SASH1(through either the overex-pression or mutation of SASH1)showed a pattern of heteroge-neous expression(Fig.S2-b4to Fig.S2-e4).The heterogeneous pattern of SASH1in vitro was also observed in vivo(Fig.1C).In addition,most of the SASH1-positive cells were melanocytes that were nucleic positive for Mitf,a melanocyte marker,andª2016The Authors.Journal of Cellular and Molecular Medicine published by John Wiley&Sons Ltd and Foundation for Cellular and Molecular Medicine.805J.Cell.Mol.Med.Vol21,No4,2017Fig.1Mutations in SASH1increase SASH1expression in vitro and in vivo.(A)Mutation sites in the SASH1gene in three families with DUH.(B) Western blotting demonstrated the differential and increased expression of mutant SASH1proteins compared to that of wild-type SASH1in different A375cells.(C)HE staining,SASH1and Mitf immunohistochemical analysis of the epidermal tissues from the Y551D-mutation DUH-affected individ-uals and normal controls.Heterogeneous expression of the SASH1protein was observed in all of the epithelial layers in the epidermal tissues from the Y551D-mutation DUH-affected individuals as compared with that of normal controls(NC).Heterogeneous distribution of melanocytes was detected in the epithelial layers of DUH-affected individuals using the melanocyte marker Mitf as compared with that of normal controls.409magni-ﬁcation.Scale bar=20l m.Red arrows denote the representative positive cells of SASH1and Mitf.806ª2016The Authors.Journal of Cellular and Molecular Medicine published by John Wiley&Sons Ltd and Foundation for Cellular and Molecular Medicine.demonstrated a heterogeneous distribution of melanocytes in the epithelial tissues of DUH-affected individuals as compared with those of unaffected individuals.Some cytoplasm-positive staining of Mitf is false positive (Fig.1C).The phenomenon thatmelanocytes or SASH1-positive epithelial cells localized not only to the basal layers but also to the suprabasal layers of the affected epidermal tissue is coincide with our previous conclusion that SASH1mutations promote melanocyte migration[11].Fig.2G a s interacts with SASH1and is a pivotal downstream of p53/POMC cascade.(A )The associations between GFP-SASH1and endogenous G a s were identiﬁed by immunoprecipitate-Western blot (IP-WB)analysis in HEK-293T cells.HEK-293T cells were transfected with the pEGFP-C3-SASH1vectors.At 24hr post-transfection,GFP-SASH1was immunoprecipitated (IP),and the associated GFP-SASH1was detected by Western blot analysis using an anti-GFP antibody.Different sizes of G a s bands were observed,at 28,46,68and 111kD,which may be caused by post-transla-tional modiﬁcations (PTMs).(B )GFP-G a s is associated with Flag-SASH1.HEK-293T cells were co-transfected with the pEGFP-C3-G a s and pBABE-puro-Flag-SASH1vectors.At 36hr post-transfection,Flag-SASH1was immunoprecipitated,and the associated GFP-G a s was detected by Western blot analysis using an anti-GFP antibody.(C )and (D )P53,POMC and SASH1is necessary for the activation of G a s.HEK-293cells and NHEMs were transfected with HA-p53,myc-POMC and GFP-SASH1,respectively,according to different manners of combination.After 36hr after transfection,two normal cells were lysed and subjected to immunoblotting with GAPDH as loading control.(E )Exogenous G a s is induced by p53.HA-p53and GFP-G a s were introduced into HEK-293cells.After 36hr after transfection,cells were lysed and subjected to immunoblotting.Exogenous G a s was activated by gradually increased amounts of exogenous p53(HA-p53).(F )Exogenous G a s is induced by SASH1.GFP-G a s and GFP-SASH1were introduced into HEK-293T cells.Exogenous G a s was induced by gradually increased doses of exogenous SASH1.(G )and (H )Exogenous p53(HA-p53)overexpression induces exogenous POMC(myc-POMC)expression in a dose-dependent manner in HEK-293T cells and NHEMs.Different dose of HA-p53vector and a certain amounts of myc-POMC vector were transfected into HEK-293T cell for expression.Exogenous POMC RNA levels were measured by quantitative RT-PCR and normalized to GAPDH.Results of RNA levels are expressed as the mean of the experiment carried out in triplicate Æthe S.D.The expression of HA-p53and myc –POMC was analysed by Western blot as GAPDH as loading control.ª2016The Authors.Journal of Cellular and Molecular Medicine published by John Wiley &Sons Ltd and Foundation for Cellular and Molecular Medicine.807J.Cell.Mol.Med.Vol 21,No 4,2017SASH1is associated with G a s and induced by the canonical p53/POMC/G a s cascadeThe functional domains of SASH1(SAM and SH3)suggest that this protein plays a role in a signalling pathway as a signalling molecule adapter or as an associated scaffolding protein[8,9].Therefore,we performed a pull-down assay and a mass spectrometry analysis to investigate which signalling pathways are activated by SASH1.The pull-down assay and LC-MS/MS analysis demonstrated that SASH1 interacts with G a s and CALM,both of which are important in melano-genesis process(Table S1)in WT-A375cells.G a s is a key element of the classical signal transduction pathway linking receptor-ligand inter-actions with the activation of adenylyl cyclase and a variety of cellular responses[17].To investigate the associations between SASH1and G a s,HEK-293T cells were co-transfected with Flag-SASH1and GFP-G a s.Exogenous SASH1was immunoprecipitated with both exoge-nous G a s(GFP-G a s)and endogenous G a s.Exogenous SASH1 immunoprecipitates had different observed band sizes of G a s (Fig.2A and C).G a s mediates cAMP production in melanocytes which is stimu-lated by a-MSH and melanocortins[18],and our study here shows that G a s is associated with SASH1.Hence,we examine whether G a s is required for the induction of SASH1and how G a s mediates SASH1 expression,we introduced exogenous p53,POMC,G a s and SASH1 gene into HEK-293T and NHEMs to assess the effects of p53and POMC on G a s.Exogenous G a s was induced in the co-existence of exogenous p53and POMC(Fig.2C lane5and Fig.2D lane5),and both inducements of exogenous G a s and exogenous SASH1were observed in the co-existence of exogenous p53and POMC in two types of normal cells(Fig.2C lane6and Fig.2D lane6).Mean-while,in the presence of GFP-SASH1,GFP-G a s was also induced (Fig.2C lane4and Fig.2D lane4),which indicated that SASH1is necessary for the activation of GFP-G a s.And immunoblot showed that G a s was identiﬁed to be induced by exogenous p53and SASH1(Fig.2E and F).Our results also demonstrated that POMC was mediated by p53in HEK-293T and melanocytes were consis-tent with previous conclusions[1](Fig.2G and H).Conversely, endogenous SASH1and exogenous SASH1were induced by G a s (Fig.3A and B).To conﬁrm the fact that POMC,p53and G a s are necessary for the inducement of SASH1,exogenous POMC,p53,G a s and SASH1were transfected into HEK-293T cells and followed by silence of G a s and POMC by two speciﬁc pairs of siRNA,respectively.As identiﬁed in HEK-293cells,knockdown of G a s gene directly induced signiﬁ-cant reduction in SASH1(Fig.3C and D).Silencing of POMC resulted in the down-regulation of G a s and SASH1(Fig.3E and F).Taken above,it is believed that G a s serves as a pivotal downstream of p53/ POMC cascade and SASH1is regulated by a novel p53/POMC/G a s cascade.SASH1is physiologically induced by p53upon UV stimulationTo verify that SASH1is induced physiologically by p53,discarded normal human foreskin specimens were exposed to gradually increased dose of UV and stained for the histological analyses of p53, ACTH/POMC and SASH1.Immunohistochemical(IHC)analyses revealed p53is rapidly induced in basal layers at the0.5J/cm2dose of UV irradiation.The rapid induction of SASH1and POMC/ACTH at 1.0J/cm2dose of UV irradiation in melanocytes is followed by p53 up-regulation(Fig.4A).Previous reports had suggested that the up-regulation of POMC gene is induced at both protein and mRNA levels following UV irradiation of skin[19,20].Followed the previous descriptions[1],a100J/m2UVB dose was administered in this experiment.This dose is equivalent to the standard erythema dose (SED),which is commonly used as a measure of sunlight[21].So HEK-293T cells and NHEMs were transfected with exogenous POMC and followed by UV irradiation,both endogenous p53and SASH1pro-tein levels were assessed by immunoblot.UV markedly induced expression of exogenous POMC and endogenous SASH1by6hr,and p53induction was already maximal by3hr,which is consistent with its known stabilization by UV in NHEMs.At24hr,the levels of POMC, p53and SASH1protein were maximally induced by UV in NHEMs (Fig.4B).Similar inducement of exogenous POMC and endogenous p53and SASH1by UV irradiation was observed in HEK-293T cells (Fig.4C).Hence,we believe that not only POMC but also SASH1acts as a novel downstream partner which is responsive to the induction of p53by UV irradiation.Reciprocal induction between p53and SASH1is induced in normal cellsTo examine whether p53is required for the induction of SASH1, we introduced exogenous p53and POMC gene into HEK-293T and NHEMs to assess the induction of p53and POMC to SASH1. Exogenous SASH1was induced by p53in the presence of POMC (myc-POMC)in NHEMs and HEK-293T cells(Fig.S3).Exogenous SASH1was induced by increasing amounts of exogenous p53in two normal cells(Fig.5A and B).Conversely,exogenous p53was promoted by increasing amounts of exogenous SASH1(Fig.5CFig.3A novel p53/POMC/G a s/SASH1cascade regulates the expression of SASH1.(A)and(B)Endogenous or exogenous SASH1is induced by G a s.Gradually increasing amounts of exogenous G a s(GFP-G a s)and exogenous SASH1(GFP-SASH1)or only different doses of exogenous G a s were transfected into HEK-293T cells.The expression of endogenous or exogenous SASH1was analysed by immunoblotting along with GAPDH as loading control.(C)and(D)G a s is necessary for the inducement of SASH1.After transfection with GFP-G a s,myc-POMC and GFP-SASH1as well as increasing doses of HA-p53according to different combinations,two groups of HEK-293cells were subsequently introduced with two pairs of effective G a s siRNAs and negative control(NC)siRNA.Protein levels were detected by immunoblot.(E)and(F)POMC is necessary for the induce-ment of SASH1and G a s.After introduction into GFP-G a s,myc-POMC and GFP-SASH1as well as increasing dose of HA-p53according to different manner of combinations,two groups of HEK-293cells were subsequently silenced with two pairs of effective POMC siRNAs and NC siRNA.808ª2016The Authors.Journal of Cellular and Molecular Medicine published by John Wiley&Sons Ltd and Foundation for Cellular and Molecular Medicine.。

siRNA转染实验指导

siRNA转染RNA干扰现象是由与靶基因序列同源的双链RNA引发的，广泛存在于生物体内的序列特异性基因转录后的沉默过程。

本次实验采用罗氏的X-tremeGENE siRNA转染试剂进行实验。

一、细胞准备转染前一天，取出处于对数生长期的细胞，吸掉原来的培养液，用无菌PBS清洗细胞。

加入1ml胰酶消化液消化细胞，显微镜下观察所有细胞完全皱缩变圆后加入终止液终止消化。

收集细胞悬液于15ml离心管中，800r/min室温离心5min。

用罗氏CASY快速细胞计数及活率分析仪进行细胞计数。

弃去上清，加入无抗生素培养基重悬细胞。

以1~4×105细胞/孔的密度接种到6孔培养板上，无抗生素培养基培养一天。

细胞转染前需用高质量血清进行培养，使细胞健康，否则将造成细胞营养不良，细胞呈现瘦削拉长状态，也会影响转染效率。

转染前3小时，取出无抗生素培养的细胞置于倒置显微镜下观察。

若融合率达50％～70％，即可进行转染实验。

二、耗材及试剂准备实验开始前将枪头、去核酸酶的EP管、试管架等放入无菌操作台，以紫外线照射30min。

转染前温育Optimen I Reduced Serum Medium，以及将X-tremeGENE siRNA转染试剂、siRNA 置于15-25度室温条件下平衡。

取出转染试剂，对EP管做好标记。

siRNA小干扰RNA 转染程序

siRNA小干扰RNA 转染程序将制备好的siRNA和其表达载体转导至真核细胞中的方法主要有以下几种：磷酸钙共沉淀、电穿孔法、DEAE-葡聚糖和polybrene、机械法（显微注射和基因枪）、阳离子脂质体试剂，其中阳离子脂质体试剂转染法是目前最常用的转染方法。

目录一、转染方法哺乳动物转染的常见方法有：磷酸钙共沉淀、电穿孔法、DEAE-葡聚糖和polybrene、机械法（例如，和基因枪）、阳离子脂质体试剂，其中阳离子脂质体试剂转染法是目前最常用的转染方法。

二、脂质体型转染(一)注意事项1.转染试剂的用量2.si（小干扰）的用量3.转染时的细胞密度4.转染时的操作顺序5.细胞与转染试剂/si（小干扰）复合物的温浴的时间(二)SiMi转染试剂选择最适合的转染试剂和转染条件，往往取决于不同的哺乳动物细胞类型和不同的核酸分子.SiMiTransfectionReagents适用于核酸的体内和体外操作，可应用于DNA、、反义寡核苷酸、si（小干扰）的转染，也可应用于DNA/si（小干扰）的共转染操作；是一种新型的高效si 转染试剂。

(三)SiMi应用领域1.原代培养细胞和细胞株的基因转染2.si（小干扰）高通量转染试验3.DNA转染；DNA和si（小干扰）的共转染4.核酸（si、DNA、）的体内导入试验5.贴壁细胞和悬浮细胞转染(四)SiMi特点1.不必更换培养基，操作简便易行，可在半小时内完成操作2.在含血清培养基中也能表现高转染效率3.细胞毒性低；适用细胞广泛4.即用型试剂，可在含有抗生素的完全培养基中转染5.基于脂质的转染试剂，确保没有se活性6.可介导si（小干扰）高转染细胞及体内si（小干扰）的高效导入三、适用细胞类型SiMiTransfectionReagents转染试剂可广泛应用于多种细胞系的DNA和si（小干扰）转染如：HeLa(人颈部癌细胞)、MCF-7(人乳房癌细胞)、Hep3B(人肝细胞癌细胞)、COS-7(猴肾细胞)、Neuro-2a(鼠神经母细胞瘤细胞)、NIKS(人角质化细胞)、B16(鼠黑素瘤细胞)、DLD-1(人结肠癌细胞)、NIH/3T3(鼠胚胎成纤维细胞)、HT-29(人结肠腺癌细胞)、A549(人肺癌细胞)、CHO-k1(仓鼠卵巢细胞)和293(腺病毒5DNA的人胚胎肾细胞)，SVRbag4细胞等。