Figure 3
From: Downregulation of SAV1 plays a role in pathogenesis of high-grade clear cell renal cell carcinoma
Knockdown of SAV1 induces cell proliferation and inhibits apoptosis. a Expression of SAV1 protein was suppressed in HK2 cells and RPTEC transfected with specific siRNAs. Two independent Stealth™ RNAi oligonucleotides for SAV1 (SAV1-siRNA1 and SAV1-siRNA2), or the Stealth™ RNAi Negative Control Duplex siRNAs (control-siRNA) were transfected into HK2 and RPTEC cells. After 72 h, protein was extracted and analyzed by Western blotting with anti-SAV1 antibody and anti-GAPDH antibody. SAV1 was markedly downregulated in HK2 and RPTEC cells transfected with SAV1-siRNA1 and SAV1-siRNA2. b Knockdown of SAV1 induces proliferation of HK2 cells and RPTEC. Upper column; MTS assay at 72 h after transfection. Y axis indicates the absorbance at 492 nm for MTS assay. Experiments were performed in triplicate, and bars indicate s.d.. * p < 0.05; Student's t test. Lower column; BrdU incorporation was estimated as the absorbance of cells transfected with SAV1-siRNA1 and SAV1-siRNA2, and those transfected with control siRNA. Experiments were performed in triplicate, and bars indicate s.d.. * p < 0.05; Student's t test. MTS assay as well as BrdU incorporation demonstrated that proliferation of HK2 cells and RPTEC transfected with SAV1-siRNA1 and SAV1-siRNA2 was significantly increased relative to cells transfected with control siRNA. c Apoptotic activity after transfection with SAV1 siRNAs was significantly inhibited. Upper column; HK2 cells transfected with control siRNA, SAV1-siRNA1 and SAV1-siRNA2 were incubated for 72 h and then analyzed using a cell death ELISA kit. Y axis indicates the relative ratio of the absorbance at 492 nm for control cells set at 1.0. The results are shown as means ± s.d. of three independent experiments. * p < 0.001; Student's t test. Lower column; HK2 cells transfected with control siRNA, SAV1-siRNA1 and SAV1-siRNA2 were incubated for 72 h and then analyzed using a Caspase-Glo 3/7 Assay kit. Y axis indicates the relative ratio of the fluorescent signal for control cells set at 1.0. The results are shown as means ± s.d. of three independent experiments. *p < 0.001; Student's t test.