Fig. 2

Macropinocytosis-mediated acquisition of cysteine rescues sorafenib-induced ferroptosis. A Gene set enrichment analysis of published ferroptosis expression signatures among genes expressed by sorafenib-treated SK-Hep1 cells. B Representative immunofluorescence staining with anti-4-HNE (left panel) and quantification of 4-HNE fluorescence in response to sorafenib in tumor tissues from ten treatment-naïve HCC patients (right panel). C Representative images of C11-BODIPY, a marker of lipid peroxidation, in SK-Hep1 cells treated with sorafenib, either alone or in combination with the antioxidant GSH or β-mercaptoethanol (β-ME). C11-BOIPY represents levels of staining with the probe (unoxidized), while oxC11-BODIPY (oxidized) indicates the levels of lipid ROS. D Representative images of C11-BODIPY in SK-Hep1 and Huh7 cells treated with sorafenib, either alone or in combination with BSA and/or EIPA (upper panel). Quantification of C11-BODIPY fluorescence in cells (lower panel). E Genes shown in the heatmap of SK-Hep1 cells treated with sorafenib, either alone or in combination with BSA or/and EIPA. F Relative cysteine levels in SK-Hep1 and Huh7 cells treated with sorafenib, either alone or in combination with BSA, EIPA, and/or bafilomycin A1, an inhibitor of protein degradation in lysosomes. G Relative GSH levels in SK-Hep1 and Huh7 cells treated with sorafenib, either alone or in combination with BSA and/or EIPA. H Effect of sorafenib on CBS proteins in control siRNA or siCBS-transfected SK-Hep1 and Huh7 cells. I Representative images of C11-BODIPY in SK-Hep1 and Huh7 cells shown in Fig. 2H. Data are normalized against values measured in vehicle-treated cells (Con) and expressed as the mean ± SEM of at least three independent experiments. Scale bar, 20 µm. *p < 0.05, **p < 0.01, and *** p < 0.001