Effect of RTA 405 on survival in cell lines with low, moderate, or high basal NRF2 activity.

A. IC₅₀ values for cell lines treated with RTA 405 for 48 hours. IC₅₀ values for 2 cell lines (SK-MEL-5 and SK-N-SH) could not be determined using the tested concentration range and are excluded from the graph. B. GI₅₀ values for cell lines treated with RTA 405 for 72 hours. C. Maximum RTA 405-induced caspase-3/7 activity observed in cells treated with 1600 nM RTA 405 for 24 hours. Caspase-3/7 activity was normalized to activity in 786–0 cells (value, 100). D. Cells with low (L), moderate (M), or high (H) basal NRF2 activity were treated with RTA 405 for 24 hours and levels of caspase-3, caspase-9, cyclin D1, CDKN1A (p21), XIAP, and BIRC2 were evaluated by western blot in whole-cell lysates. Actin served as a loading control. E. Cells were mock transfected or transfected with non-targeting or NRF2 siRNA and then treated with RTA 405 for 72 hours. Growth was assessed using the SRB assay. F. Effect of 125 nM RTA 405 on NQO1 mRNA levels. Cells were treated with RTA 405 for 18 hours and NQO1 mRNA levels were assessed by qPCR. G. Cells were treated with 100 nM RTA 405 for 48 hours and cell viability was determined. Percent of vehicle-treated cell survival is shown. For (A, B, G) cell viability was determined using the SRB assay. For (A-C, F, G) data points for individual cell lines are the mean of three individual experiments and horizontal lines are the mean of all cell lines in each group. Statistical significance was determined by Mann-Whitney test. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.