Figure 6.

USP32 expression confers resistance to YM155-mediated DNA damage. (A) Immunofluorescence analysis of γH2AX foci formation in different cell lines treated with 25 nM YM155 for 24 h. (B) A quantification graph from (A) showing the percentage of the γH2AX-positive population in different cancer cell lines. Data are presented as the mean and standard deviation of three independent experiments. Scale bar = 50 µm (C) Intracellular uptake of YM155 drug levels was determined by multiple-reaction monitoring-mass spectroscopy analysis (MRM-MS) in MCF7 and PC3 cells exposed to 3 µM YM155 for 90 min. Data are presented as the mean and standard deviation of three independent experiments. A two-tailed t test was used and the P value is as indicated. (D-E) USP32 plasmids were dose-dependently transfected in PC3 (D) and SW480 (D) cell lines with high levels of endogenous expression of SLC35F2. Western blot analysis was performed with the indicated antibodies. GAPDH was used as a loading control. (F-G) Cells from (D) and (E) were subjected to YM155 treatment (25 nM) for 24 h and stained with EthD-1 to quantify the EthD-1-positive cells. Data are presented as the mean and standard deviation of three independent experiments. One-way ANOVA followed by Tukey's post hoc test was used and the P values are as indicated. (H-I) Validation of the single cell-derived USP32 knockout clones in MCF7 (H) and BT474 (I) cell lines using western blots with the indicated antibodies.