Fig. 6. Chemerin mitigates ferroptosis by upregulating SLC7A11 expression in renal tubular epithelial cells.

A Knockdown of SLC7A11 by siRNA. TCMK-1 cells were transfected with siRNA targeting Slc7a11 (si-SLC). Seventy-two hours post-transfection, SLC7A11 expression was analyzed by western blot, with β-Actin used as a loading control. n = 3 (independent experiments). B Quantification of SLC7A11 expression from the western blots shown in (A). C SLC7A11 knockdown reverses the changes in GPX4 and ACSL4 induced by chemerin. TCMK-1 cells were transfected with si-SLC for 48 h, then treated with erastin (Era, 5 μM) and the recombinant chemerin (20 ng/ml) for an additional 24 h. Protein expression was analyzed by western blot, with β-Actin used as a loading control. n = 3 (independent experiments). D Quantification of GPX4 and SLC7A11 from the western blots shown in (C). E Reactive oxygen species (ROS) levels. Cell treatments were as described in (C). n = 5 (independent experiments). Scale bar = 50 μm. F Quantified ROS levels from the images shown in (E). G Calcein/PI staining. Cell treatments were as described in (C). n = 5 (independent experiments). Scale bar = 50 μm. H Quantified PI-positive cells from the images shown in (G). I ATP levels. Cell treatments were as described in (C). n = 3 (independent experiments). J Lipid peroxidation. Oxidized lipids were captured by using C11 BODIPY and assayed by flow cytometry. Cell treatments were as described in (C). n = 3 (independent experiments). K Quantified lipid peroxidation from the images shown in (J). Lipid hydroperoxide (LPO; L), malondialdehyde (MDA; M), cystine uptake (N), and GSH levels (O). Cell treatments were as described in (C). n = 3 (independent experiments). Data are presented as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, by one-way ANOVA with Tukey’s multiple comparison test.