Fig. 1. RSL3-induced ferroptosis of human renal tubular cells. (A) HKC-8 cells were treated with 1 µM RSL3 (6 h), 10 µM erastin (24 h), and 1 µM staurosporine (STS 2 h). Cleaved caspase-3 and cleaved PARP expressions were analyzed by western blotting. After HKC-8 cells were treated with RSL3 and inhibitors for 6 h, cell death was measured using Alamar blue assay. Fer-1: 1 µM ferrostatin-1; DFO: 100 µM deferoxamine; Z-VAD: 20 µM Z-VAD-FMK; Nec-1: 2 µM necrostatin-1; NAC: 5 mM N-acetyl cysteine. (B) HKC-8 and HK-2 cells were treated with RSL3 (1 µM) and ferroptosis inhibitors for 12 h; thereafter, the cell viability was assessed using the Alamar blue assay. (C) HKC-8 cells were treated with RSL3 (1 µM) and ferroptosis inhibitors and then stained with SYTOX green for 12 h. SYTOX green stains ferroptosis-induced cells. Scale bar=50 μm. (D) HKC-8 cells were treated with RSL3 (1 µM) and ferrostatin-1 (1 µM) for 1 h and then stained with BODIPY C11 for 30 min. BODIPY C11 stained cells were analyzed using a CytoFLEX Flow Cytometer. Data are represented as the mean ± SD for three separate experiments (*p<0.05, **p<0.01 and ***p<0.001).
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