Figure 1.

USP10 is a new component of the TGF‐β pathway. (A) Depletion of endogenous Smad4 with shRNAs markedly decreased cell migration by transwell migration assays. Bel‐7402 cells infected with lentivirus encoding the indicated shRNAs were treated with TGF‐β (5 ng·mL⁻¹) for 24 h. (B) Depletion of endogenous Smad4 with shRNAs significantly inhibits TGF‐β transcriptional activity. 293T cells infected with lentivirus encoding Smad4 shRNAs were transfected with PGL4.14‐SBE₄‐luc. Cells were starved without serum overnight and then treated with TGF‐β (10 ng·mL⁻¹) for 6 h. (C) A cell‐based luciferase‐screening model to screen the potential USPs regulating TGF‐β signaling by using siRNAs to inhibit the expression of 54 known or predicted human USPs. (D) Eight USPs siRNA pools significantly inhibited TGF‐β responses. Pooled USPs siRNAs (a mix of oligos for each gene, 50 nm) and PGL4.14‐SBE₄‐luc reporter were transfected in 293T cells, and cells were starved without serum overnight and then treated with TGF‐β (10 ng·mL⁻¹) for 6 h. (E, F) Depletion of USP10 minimizes Smad4 protein levels. HepG2 cells were infected with lentivirus encoding the indicated shRNAs. Cell lysates were immunoblotted with indicated antibody and 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) The depletion of endogenous USP10 with three independent shRNAs targeting different coding regions of USP10 indeed dramatically inhibits TGF‐β transcriptional activity. 293T cells infected with lentivirus encoding the indicated shRNAs were transfected with PGL4.14‐SBE₄‐luc. Cells were starved without serum overnight and then treated with TGF‐β (10 ng·mL⁻¹) for 6 h. The results represent the means (±SD) of three independent experiments. ***P < 0.001.