Fig. 8

Fig. 8

a Cells were pre-incubated for 1 h in presence or absence of 50 mM NAC or 60 µM necrostatin (Nec)−1. After 4 and 8 h of treatment with TMQ0153 at 20 and 30 µM, reactive oxygen species (ROS) levels were measured by flow cytometry following dichlorofluorescein diacetate (H₂DCFDA) staining. H₂O₂ was used as a positive control for ROS induction. b Quantification of total GSH levels (left panel) and GSH (glutathione)/glutathione disulfide (GSSG) ratio (right panel). 50 µM Buthionine sulfoximine (BSO) was used as a positive control for the inhibition of GSH synthesis. c Cells were stained with Hoechst and Lysotracker Red and analyzed by fluorescence microscopy. Lysotracker Red fluorescence intensity was quantified using Image J 1.8.0 software (upper panel). Lysotracker Red intensity was quantified by FACS (bottom panel). Chloroquine (CQ; 75 μM, 4 h), PP242 (PP, 10 μM, 4 h) and baf-A1 (40 nM, 4 h) were used as a positive and negative controls for autophagy inhibition and induction, respectively. All pictures are representative of three independent experiments and data represent the mean (±S.D.) of three independent experiments. Statistical significance was assessed as *p < 0.05, **p < 0.01, ***p < 0.001 compared to untreated cells unless otherwise specified. One-way ANOVA (LMP); post hoc; Dunnett’s test. One-way ANOVA (ROS, GSH assay); post hoc; Tukey’s test.