Fig. 6: Elevation of ROS by cholesterol depletion is required for JNK activation and for the effects on LC3-II and p62 levels.

Mv1Lu cells in 6-well plates were subjected (or not) to CD by statin (16 h) with or without the ROS scavenger NAC (10 mM; A–E), or the JNK inhibitor SP600125 (20 μM; F–H). A CD elevates ROS. Cells were treated (or not) with H₂O₂ (0.75 mM, 2 h; positive control), and incubated at 37 °C with DCF (10 μM, 30 min). After washing, DCF fluorescence intensity was determined by FACS with excitation at 485 nm and emission at 525 nm, taking the intensity measured in untreated cells in the same experiment as 100%. Bars are mean ± SEM of 3–7 independent experiments. Asterisks indicate a significant difference between the indicated pairs (**P < 0.01; ***P < 0.001; one-way ANOVA and Bonferroni post hoc test). B–E The ROS scavenger NAC prevents the effects of CD on JNK activation and on LC3-II and p62 levels. A representative blot is shown in panel B. Quantification of the effect of NAC on the levels of the proteins tested is depicted for pJNK (C), LC3-II (D), and p62 (E). The control sample (no CD, no NAC) for each protein was taken as 1. Bars are mean ± SEM of 5 independent experiments (*P < 0.05; Student’s two-tailed t-test). F–H Inhibition of JNK activity prevents the effect of CD on LC3-II and p62 levels. F A representative blot. G, H Quantification of the effects on LC3-II and p62 protein levels. Bars, mean ± SEM of 5 independent experiments. In each blot, the untreated sample (control) was taken as 1. Asterisks indicate significant differences between the pairs of samples indicated by the brackets (*P < 0.05; Student’s two-tailed t-test).