Fig. 6.

MDM2 inhibitor enhanced the antipancreatic cancer effects of USP22 overexpression. (A,B) RT‐PCR (A) and western Blot (B) analysis were used to detect the expression of p21 in SW 1990 cells. GAPDH served as an internal reference and repeated for three replicates. Statistical analyses were performed with one‐way ANOVA followed by Tukey's multiple comparison's tests. ns, not significant; *P < 0.05; ***P < 0.001. (C–E) SW 1990 cells infected with or without USP22 plasmid and treated with or without MDM2 inhibitor were harvested for MTS (C), apoptosis (D), and cell cycle assays (E). Each bar represents the mean ± SD of three independent experiments. Statistical analyses were performed with one‐way ANOVA followed by Sidak's multiple comparison's tests. *P < 0.05; **P < 0.01 ***P < 0.001. (F–H) SW 1990 cells infected with or without USP22 plasmid were subcutaneously injected into nude mice and with or without MDM2 inhibitor. The tumors were harvested and photographed (F) on day 21. Data for tumor volume (G) and tumor mass (H) are shown as the mean ± SD (n = 5). Statistical analyses were performed with two‐way ANOVA followed by Sidak's multiple comparison's tests. ***P < 0.001. (I) Mouse tumors were harvested for western blot analysis to detect the expression of p21 and caspase 3 (to determine the apoptosis level) in the tumors. GAPDH served as an internal reference and repeated for three replicates. (J) Mouse tumor sections were stained with Ki‐67 antibody to determine cell proliferation level. Data are shown as means ± SD (n = 5). The scale bar indicated 50um. Statistical analyses were performed with one‐way ANOVA followed by Tukey's multiple comparison's tests. ***P < 0.001. p21: Cyclin‐dependent kinase inhibitor 1A, also symboled as CDKN1A; GAPDH: Glyceraldehyde‐3‐phosphate dehydrogenase.