Research Paper Volume 11, Issue 21 pp 9778—9793

Figure 4. USP5 knockdown induced p27 expression via repressing HDAC2 expression. (A) OVCAR3 cells were transiently infected with USP5 shRNAs (#1 and #2), control shRNA (NC) or untreated (Control). Real-time PCR analysis of p16, p21, p27, RBL2, CCND1 and c-Myc. (B) GSEA analysis in ovarian cancer patients with higher USP5 expression versus lower USP5 expression (TCGA dataset). NES, normalized enrichment score. (C) Western blot analysis of HDAC Class I members and p27. (D) Western blotting and real-time PCR analyses were performed to check the expression of HDAC2 and p27 in CAOV3 cells expressing NC or USP5 shRNAs (#1, #2). (E) The protein expression of USP5, HDAC2 and p27 in xenografts formed from OVCAR3 cells expressing control (NC) or USP5 shRNA (#1). Three samples were randomly chosen from each group. (F-H) SKOV3 cells were infected with USP5 overexpressing virus (USP5 OE) or control virus (Vector), and treated with HDAC2 siRNA (siHDAC2) or control siRNA (siNC) as indicated. The protein expression of USP5, HDAC2 and p27 was detected, and mRNA expression of p27 was evaluated at 48 h after treatment (F). Proliferation (G) and cell cycle distribution (H) was evaluated by CCK-8 and PI/flow cytometry analysis, respectively. *P<0.05,**P<0.01, ***P<0.001 vs. Vector+siNC; $$P<0.01, $$$ P<0.001 vs. USP5 OE+siNC (ANOVA test followed by a Tukey post hoc test).