Fig. 3: Par-4 regulates ferroptosis through autophagy.

a Indicated cells were treated with RSL3 (2 μM) and erastin (10 μM) for the indicated time period. Following the treatment, Western blot analysis of LC3 and p62/SQSTM1 were detected. The relative density of protein bands were quantified and normalized to the actin of each group, and fold changes were presented in histogram from three independent experiments. Indicated cells were treated with RSL3 (2 μM) and erastin (10 μM) for 24 h in the presence or absence of BafA1 (250 nM) or HCQ (5 μM). Following the treatment, b Cell viability by MTT assay. Data shown are mean ± SD; n = 3 samples, and c Western blot analysis of indicated proteins were measured. The relative density of protein bands were quantified and normalized to the actin of each group, and fold changes were presented in histograms from three independent experiments. d U87MG cells expressing ptfLC3 were treated with RSL3 (2 μM) for 6 h. Representative fluorescent images were captured at x20 magnification using fluorescent microscopy. BafA1 (250 nM) was used as a negative control for autolysosome formation. Yellow puncta represent the number of autophagosomes, and red puncta in merged images represent autolysosomes. Indicated cells were treated with RSL3 (2 μM) for 3 h and erastin (10 μM) for 24 h in the presence or absence of BafA1 (250 nM) or HCQ (5 μM). Following the treatment, e Intracellular labile iron was determined using flow cytometry. The bar graph showing labile iron level was expressed as a percentage of the control, and f Lipid peroxidation was detected by flow cytometry. Bar graph showing the relative levels of lipid peroxidation. Data shown are mean ± SD; n = 3 samples. g U87MG cells were stably transfected with Atg-5-shRNA or Atg-7-shRNA, followed by exposure to RSL3 (2 μM) for 3 h. Following the treatment, a Western blot analysis of indicated proteins were performed. The relative density of protein bands were quantified and normalized to the actin of each group, and fold changes were presented in histograms from three independent experiments. U87MG cells were transiently transfected with siAtg-5 and siAtg-7, followed by exposure to RSL3 (2 μM) for 3 h. Following the treatment, h intracellular labile iron was determined using flow cytometry. The bar graph showing labile iron level was expressed as a percentage of the control, and i Lipid peroxidation was detected by flow cytometry. Bar graph showing the relative levels of lipid peroxidation. Data shown are mean ± SD; n = 3 samples. j U87MG cells were stably transfected with Atg-5-shRNA or Atg-7-shRNA. After transfection, cells were treated with RSL3 (2 μM; Atg-5-shRNA and 0.5 μM; Atg-7-shRNA) for 24 h. Following the treatment, cell viability by MTT assay was assessed. Data shown are mean ± SD; n = 3 samples. U87MG cells were stably transfected with k Par-4-shRNA and l Par-4-CRISPR. After transfection, cells were treated with RSL3 (2 μM) for 3 h, and a Western blot analysis of indicated proteins was carried out. The relative density of protein bands were quantified and normalized to the actin of each group, and fold changes were presented in histograms from three independent experiments. m A172 cells were stably transfected with Par-4-shRNA. After transfection, cells were treated with erastin (10 μM) for 24 h, and a Western blot analysis of indicated proteins was carried out. The relative density of protein bands were quantified and normalized to the actin of each group, and fold changes were presented in histograms from three independent experiments. n U87MG and o A172 cells were transiently transfected with pCMV6-DDK-Par-4. After transfection, cells were treated with RSL3 (2 μM) for 3 h. Following the treatment, a Western blot analysis of indicated proteins were carried out. The relative density of protein bands were quantified and normalized to the actin of each group, and fold changes were presented in histograms from three independent experiments. Data shown are mean ± SD; n = 3 samples. Statistical significance (P values) was analyzed by one-way ANOVA using the Bonferroni post-hoc test.