Figure 2.

USP10 positively regulates Smad4 protein levels in HCC. (A, B) Knockdown of USP10 down‐regulates Smad4 protein levels, but had little effect on Smad2/3 protein levels. HepG2 and Bel‐7402 cells were infected with lentivirus encoding the indicated shRNAs, and cell lysates were immunoblotted with indicated antibody. (C, D) Overexpression of USP10‐WT, but not USP10‐C424A, up‐regulates the protein levels of Smad4, but not that of Smad2/3. HepG2 and Bel‐7402 cells transfected with indicated constructs were lysed, and cell lysates were immunoblotted with indicated antibody. (E) USP10 inhibitor Spautin‐1 significantly decreased Smad4 protein levels. Bel‐7402 cells were treated for 24 h with 10 μm Spautin‐1, and cell lysates were immunoblotted with indicated antibody. (F) Knockdown of USP10 had little effect on Smad4 mRNA levels. HepG2 and Bel‐7402 cells infected with lentivirus encoding the indicated shRNAs were subjected to qRT‐PCR to examine the indicated mRNA levels. Transcript levels were determined relative to GAPDH mRNA levels and normalized relative to control cells. (G, H, I) Knockdown of USP10 dramatically decreased Smad4 protein stability, but not that of Smad2/3. HepG2 and Bel‐7402 cells stably expressing the indicated shRNAs were treated with cycloheximide (CHX, 40 μg·mL⁻¹) for the indicated times; then, proteins were extracted and subjected to western blot to examine the indicated protein levels. The results represent the means (±SD) of three independent experiments. ^n.s. P > 0.05, * P < 0.05, **P < 0.01, ***P < 0.001.