Figure 5. USP5 regulated HDAC2 ubiquitination. (A) OVCAR3 cells were infected with USP5 shRNA (#1) or control shRNA (NC) in the presence and absence of 10 μM MG132 for 48 h. The protein expression of HDAC2 was assessed. (B) Cell lysates from OVCAR3 cells were immunoprecipitated (IP) with anti-HDAC2 or anti-USP5 or IgG, and then western blotting analysis was performed with anti-HDAC2 or anti-USP5. (C) Cell lysates from OVCAR3 cells infected with USP5 shRNA (#1) or control shRNA (NC) were IP with anti-HDAC2 or IgG, and western blotted with anti-ubiquitin. (D) Western blotting analysis of USP5, HDAC2 and p27 expression in ovarian cancer tissues from cohort 2 (n=16). (E) Pearson correlation scatter plots showed a positive correlation between USP5 and HDAC2, and a negative correlation between p27 and HDAC2. (F) Early apoptosis induced by PXD101 (1 μM) or cisplatin (5 μg/ml) in primary ovarian cancer cells with or without USP5 amplification. (F) 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 for 24 h. Early apoptosis induced by PXD101 (1 μM) or cisplatin (5 μg/ml) was detected at 48 h post treatment. **P<0.01, ***P<0.001 vs. Vector+siNC; $$P<0.01 vs. USP5 OE+siNC (ANOVA test followed by a Tukey post hoc test). (H) A working model that USP5 plays a key role in cell proliferation by inhibiting HDAC2 ubiquitination.