Fig 2: Lentivirus-mediated interference downregulates the expression of PPP5C in urinary bladder cancer cells. (A) T24 and BT5637 cells were examined by fluorescence microscopy after lentivirus infection for 72 h. (B and C) Knock-down efficiency of PPP5C mRNA level in T24 and BT5637 infected by Lv-shPPP5C was determined by real-time qPCR (***P<0.001). (D and E) The protein level of PPP5C was downregulated in T24 and BT5637 cells. Lv-shCon, lentivirus-mediated non-silencing shRNA; Lv-shPPP5C, lentivirus-mediated non-silencing shPPP5C. Data were presented as mean ± SD from at least three experiments.

Fig 3: PPP5C was a direct gene of miR-365b-3p. (A) Putative miR-365b-3p targeted sequences in the 3'-UTR of PPP5C mRNA are indicated. (B and C) WT or MUT 3'UTR of PPP5C and miR-365b-3p mimics or inhibitor were co-transfected into A549 or H1299 cells and the relative luciferase activity was detected by dual-luciferase reporter assay. Data were expressed as the means ± standard deviation of at least three independent experiments. **P<0.01 vs. miR-NC. (D) Protein expression of PPP5C measured in A549 cells transfected with miR-365b-3p mimics and in H1299 cells transfected with miR-365b-3p inhibitor using western blotting. (E) PPP5C protein expression measured in 95D, A549, H1299 and BEAS-2B cell lines. PPP5C, serine/threonine protein phosphatase 5; WT, wild-type; MUT, mutant; miR, microRNA; NC, negative control.

Fig 4: Apoptotic cells are increased after PPP5C silencing. (A) Apoptosis of T24 and BT5637 was analyzed in lv-shCon and lv-shPPP5C groups using flow cytometry and Annexin V/PI staining. (B) Early apoptosis (V+/PI-) and late apoptosis (V+/PI+) of the T24 and BT5637 cells were statistically analyzed (*P<0.05, **P<0.01, ***P<0.001).

Fig 5: Lentivirus-mediated knockdown of PPP5C suppresses cell viability of urinary bladder cancer cells. (A and B) After infection with Lv-shCon or Lv-shPPP5C, the growth curves of T24 and BT5637 cells were determined by MTT assay. Data are presented as mean ± SD from at least three experiments (***P<0.001). (C and D) Representative images of T24 and BT5637 cells colony formation under micro- and macro-views after infection with Lv-shCon or Lv-shPPP5C. (E and F) The number of colonies formed in T24 and BT5637 cells treated as in (C and D). Data are presented as mean ± SD from at least three experiments (*P<0.05, ***P<0.001).