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. 2018 Mar 16;7:e31275. doi: 10.7554/eLife.31275

Figure 2. SPIN1 knockdown inhibits cell proliferation and induces apoptosis.

(A) SPIN1 knockdown induces protein levels of p53 and its target genes. U2OS, H460 and HCT116p53+/+ cells were transfected with scramble siRNA (scr-siRNA) or SPIN1 siRNA and harvested 48 hr post-transfection for WB analysis with indicated antibodies. (B) SPIN1 knockdown induces mRNA levels of p53 target genes without effect on TP53 mRNA level. U2OS cells were transfected with scramble siRNA (scr-siRNA) or SPIN1 siRNA, and harvested 72 hr post-transfection for RT-qPCR (mean ± SEM, n = 2). (C) SPIN1 overexpression reduces protein levels of p53 and its target genes. HCT116p53+/+ cells were transfected with pcDNA or Flag-SPIN1 and harvested 48 hr post-transfection for WB analysis with indicated antibodies. (D) SPIN1 overexpression reduces mRNA levels of p53 target genes without effect on TP53 mRNA levels. HCT116p53+/+ cells were transfected with pcDNA or Flag-SPIN1 and harvested 72 hr post-transfection for RT-qPCR (mean ± SEM, n = 2). (E) Knockdown of SPIN1 causes p53-dependent induction of p21 and PUMA. The protein levels of p53 and its targets in HCT116p53+/+ cells and HCT116p53-/- cells that stably express scramble shRNA (scr-shRNA) or SPIN1 shRNAs were detected by WB analysis with indicated antibodies. (F) SPIN1 knockdown suppresses cell survival. HCT116p53+/+ and HCT116p53-/- cells that stably expressed scramble or SPIN1 shRNAs were seeded in 96-well plate and cell viability was evaluated every 24 hr by CCK-8 assays (mean ± SEM, n = 2). (G) Knockdown of SPIN1 inhibits clonogenic ability of colorectal cancer cells, more significantly when the cells harbor wild-type p53. HCT116p53+/+ cells and HCT116p53-/-cells that stably expressed scramble or SPIN1 shRNAs were seeded on 60 mm plates. Puromycin selection was performed for 14 days. Colonies were fixed with methanol, and visualized by staining with crystal violet (mean ± SEM, n = 3). (H) The effect of SPIN1 knockdown on apoptosis of HCT116p53+/+ cells and HCT116p53-/-cells that stably expressed scramble or SPIN1 shRNAs (mean ± SEM, n = 3). (I) U2OS cells were transfected with scramble or SPIN1 shRNA and incubated in IncuCyte S3 chamber in the presence of IncuCyte Annexin V Green Reagent for apoptosis. Positively stained cells were determined using IncuCyte analysis software. *p<0.05, **p<0.01 by two-tailed t-test (C, D, G, H,I).

Figure 2.

Figure 2—figure supplement 1. SPIN1 knockdown increase p53 and its targets protein levels in a dose-dependent manner.

Figure 2—figure supplement 1.

HCT116 p53+/+ (A) and U2OS (B) cells were transfected with a titrated concentration of SPIN1 siRNA (20 nM, 40 nM and 60 nM). And cells were harvested 48 hr after transfection and followed by WB analysis with indicated antibodies. (C) U2OS cells were co-transfected with scramble siRNA (40 nM), SPIN1-siRNA (40 nM), with or without FLAG-SPIN1 as indicated. Cells were harvested 48 hr after transfection for WB analysis.