Fig. 1: Sal triggers ferroptosis in cultured RCC cell lines.

Human proximal tubular cell line HK-2 and RCC cell lines ACHN, CAKi-1, 786-O, 769P, and OSRC2 were treated with different concentrations of Sal as indicated for 24 h. A Dose-response viability curves of RCCs and HK-2 cells treated with Sal. B Effect of 2 µM Sal against HK-2 and RCC cells measured by Sytox green-staining and C LDH release assay. D In vitro inductive effect of 2 µM Sal on lipid peroxidation measured by MDA assay. HK-2, 769P, ACHN, CAKi-1, and 786-O cells were treated with 2 µM Sal separately. E Expression of RIP3, RIP1, MLKL, and phosphorylated RIP3, RIP1, MLKL analyzed by western blot. F Caspase-3 and cleaved caspase-3 analyzed by western blot. The left panel shows the representative images, and the right panel the quantification normalized with b-actin. 786-O cells and ACHN cells were treated with 2 µM Sal for 24 h, respectively. G, H Cell viability assay in the presence or absence of the indicated inhibitors. I Flow cytometry analysis showing lipid ROS by means of fluorogenic reaction with BODIPY 589/591 C11. J Assay of ferrous ion. Data are present as mean ± S.E.M. and obtained from at least three independent experiments. P values were calculated using Student’s t-test in (I), (J), and one-way ANOVA in (B), (C), (D), (F), (G), and (H). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.