Figure 4.

Inhibition of USP9X catalytic activity depletes ZNF598 and MKRN2 protein levels. (A) HCT116 cells were treated for 4 or 24 h with a selective USP9X inhibitor (FT709, 10 µM). Cells were lysed in RIPA buffer and samples analyzed by SDS-PAGE and immunoblotted for ZNF598 and USP9X. Graph represents quantification of four independent experiments. (B) Correlation of two distinct experimental SILAC-based proteomic datasets showing the de-enrichment of ZNF598 and MKRN2 alongside known USP9X substrates (in black type) in HCT116 cells treated for 24 h with USP9X inhibitor (FT709, 10 µM). Outliers for which the ratio is either lower than Log₂(−1.0) or larger than Log₂(+1.0) in both datasets are shown in red. (C) Western blot validation of USP9X inhibitor–sensitive proteins identified in B. HCT116 cells were treated with FT709 at 5 µM (CEP131), or 10 µM (all other samples) and analyzed as in A. Graph is representative of three independent experiments. (D) De-enrichment of MKRN2 in USP9X^−/0 cells. HCT116 or HCT116 USP9X^−/0 lysates were analyzed by immunoblot with the indicated antibodies. Quantification is based on four experiments. (E) Quantitative RT-PCR reactions for MKRN2 (normalized to actin) were performed with cDNA derived from the indicated HCT116 cell lines. n = 3 independent experiments. Error bars in all panels represent standard deviation; two-tailed Student’s t test; ***, P < 0.001.