Fig. 5: H9c2 cells are sensitive to RSL3-induced ferroptosis.

a Viability of H9c2 cells treated with RSL3. H9c2 cells were treated with increasing concentrations of RSL3 for 6 h. Cell viability was measured using CellTiter-Glo. b, c Cell viability and lipid ROS analysis of H9c2 cells treated with RSL3. H9c2 cells treated with RSL3 for 6 h were subjected to a CellTiter-Glo assay (b) and an MDA assay (c). d Cell death determined by propidium iodide (PI) uptake. H9c2 cells were treated for 18 h and then treated with 10 μg/mL PI for 30 min prior to harvest. The number of PI-positive cells was determined by flow cytometry. e, f Inhibition of RSL3-induced cell death by ferroptosis inhibitors. H9c2 cells were treated with 0.5 μM RLS3 in the presence of ferroptosis inhibitors (ferrostatin-1, Fer-1; liproxstatin-1, Lip-1), an apoptosis inhibitor (zVAD-fmk, zVAD), or a necroptosis inhibitor (necrostatin-1, Nec-1) for 24 h and 6 h, and cell viability was measured. The data are the mean ± s.d.; n = 3, with ***P < 0.001 with a two-sided Student’s t-test. g Western blot analysis showing that neither apoptosis (PARP cleavage) nor necroptosis (phospho-MLKL) was activated by RSL3 in H9c2 cells. L929 cells treated with rat TNF/birinapant (T/B) or TNF/birinapant/zVAD-fmk (T/B/Z) were used as positive controls for apoptosis and necroptosis, respectively. h Prevention of RSL3-induced lipid ROS accumulation by ferroptosis inhibitors. Cells were treated with RSL3 and inhibitors as described in (f) for 6 h, and lipid peroxidation was assessed using a lipid peroxidation assay. The data are the mean ± s.d., n = 3, with **P < 0.01, ***P < 0.001 with a two-sided Student’s t-test