Figure 2.

PUM1 knockdown reduced PCa cell proliferation and survival.

A: DU145 cells were transfected with PUM1 shRNAs (shPUM1-1 and shPUM1-2 targeting different PUM1 transcript) or non-targeting shRNA (shCTL). After 48 hours, the cells were harvested and PUM1 mRNA levels were examined by qRT-PCR analysis. ACTB was used as an internal control. Data obtained from three independent experiments are presented as mean±SD. P values were calculate by two-tailed Student's t-test analysis. ^***P<0.001vs. shCTL group. B: Immunoblots showing the effect of shRNA-mediated PUM1 knockdown in DU145 and PC3 cells. C: CCK8 assay on DU145 cells transfected with the shCTL or PUM1 shRNAs as described in (A). Data are mean±SD of three independent experiments (unpaired t-test; ^***P<0.001.) D: PC3 cells were transfected with CTL or PUM1 shRNAs, and the number of viable cells at the four time points was determined using CCK8 assay. Data are mean±SD of three independent experiments. Statistical analyses were performed by two-tailed Student'st-test. ^***P<0.001. E and F: Apoptosis assay using flow cytometry after staining with annexin V-FITC/PI. Data are presented as mean±SD.^**P<0.01,^***P<0.001vs. shCTL group. G: Western blotting analysis of apoptosis-related proteins in PC3 and DU145 cells with PUM1 knockdown. H: EdU labeling of PUM1 knockdown DU145 cells revealed significantly reduced proliferation in comparison with control cells (two-tailed Student's t-test; ^**P<0.01vs. shCTL group). I: Cell cycle analysis of DU145 cells transfected with CTL or PUM1 shRNAs via flow cytometry. Data are presented as mean±SD (two-tailed Student's t-test; ^**P<0.01vs. shCTL group).