Tax stimulates ROS production in part through an interaction with USP10. (A) Jurkat cells were infected with lentiviruses encoding human USP10 shRNA (sh-USP10-1 or sh-USP10-3) or control nontargeting shRNA (sh-NT), and the cells were then cultured in the presence of puromycin. Cell lysates prepared from the selected cells were characterized using a western blot analysis with anti-USP10 and anti-α-tubulin antibodies. (B) USP10-knockdown Jurkat cells and control cells were stained with 5 μM of CM-H₂DCFDA (green) and Hoechst33258 (blue) for 5 minutes at 37°C. Staining of the cells was visualized using a fluorescence microscope. The bar indicates 10 μm. (C) The ROS levels (DCFDA-F) in cells treated as in (B) were quantitatively measured using a cell imaging software program. (D-F) USP10-knockdown (sh-USP10-1) Jurkat cells and control (sh-NT) (D) or Jurkat cells (E,F) were infected with the indicated Tax lentiviruses. The infected cells were assessed for ROS production (DCFDA-F) using 5 μM of CM-H₂DCFDA. Aliquots of the above-treated cells were subjected to a western blot analysis using anti-Tax, anti-USP10, and anti-α-tubulin antibodies. In all experiments, the values denote the mean ± SD; *P < .05; **P < .01; ***P < .001; NS indicates not statistically significant.