Fig. 2. TRIM25 facilitates ERAD and reduces ER stress induced ROS levels in tumor cells.

a Western blot analysis of the levels of CD3-δ-YFP in HCT116 cells with control (shNC) or stable knockdown of TRIM25. b, c Half-life of CD3-δ-YFP in HCT116 cells with control or stable knockdown of TRIM25, treated with cycloheximide (CHX) at the indicated times and analyzed by western blot. Representative western blot (b) and quantified graph (c) are shown. In b, the exposures of the corresponding control and TRIM25-knockdown blots were adjusted to achieve comparable levels of CD3-δ-YFP at time 0. d, e Half-life of CD3-δ-YFP in control and F-TRIM25-expressing HCT116 cells pre-treated with TG (1 μM) for 6 h, then treated with cycloheximide (CHX) at the indicated times and analyzed by western blot. Representative western blot (d) and quantified graph (e) are shown. In d, the exposures of the corresponding control and F-TRIM25-expressing blots were adjusted to achieve comparable levels of CD3-δ-YFP at time 0. f–h Flow cytometry analysis of ROS levels in control and F-TRIM25-expressing Huh7 cells treated with TM (5 μg/ml) (f) or TG (1 μM) (g) for 6 h, and quantified data is shown as (h). i Relative ROS levels in control and F-TRIM25-expressing HCT116 cells treated with TG (1 μM) for 6 h. j–l Flow cytometry analysis of ROS levels in Huh7 cells for control and stable knockdown of TRIM25 treated with TM (5 μg/ml) (j) or TG (1 μM) (k) for 6 h, quantified data is shown as (l). For c, e, h, i and l, data represent the mean ± SEM (n = 3). Statistical significance was assessed using two-tailed Student’s t-tests.