Fig. 4: Par-4 is required for the NCOA4-mediated degradation of ferritin during ferroptosis.

a Indicated cells were treated with RSL3 (2 μM) and erastin (10 μM) for the indicated time period, and then Western blot analysis of FTH1 and NCOA4 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. b A172 cells were pre-treated with DFO (50 μM) for 1 h, followed by RSL3 (2 μM) for a further 3 h. Following the treatments, a Western analysis of FTH1 was carried out. The relative density of protein bands was quantified and normalized to the actin of each group, and fold changes were presented in histograms from three independent experiments. c Indicated cells were pre-treated with DFO (100 μM) for 1 h, followed by RSL3 (2 μM) treatment for a further 3 h. After treatment, intracellular labile iron was determined using flow cytometry. The bar graph showing labile iron level was expressed as a percentage of the control. Data shown are mean ± SD; n = 3 samples. d A172 cells were treated with RSL3 (2 μM) for 3 h in the presence or absence of CPX (10 μM) or DFO (100 μM). After the treatment, the cells were subjected to FerroOrange staining to evaluate intracellular LIP. Red fluorescence signals were captured and visualized through a fluorescent microscope using constant fluorescence parameters explained in the methods section: scale bar, 50 μm. e Subsequently, median fluorescence intensity (MFI) was quantified by flow cytometry analysis. The bar graph shows relative levels of LIP by FerroOrange staining in the indicated cells. Data shown are mean ± SD; n = 3 samples. Indicated cells were treated with RSL3 (2 μM) in the presence or absence of f BafA1 (250 nM) and g Atg-5-shRNA or Atg-7-shRNA. Following the treatment, a Western blot analysis of indicated proteins were analyzed. 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 stably transfected with h Par-4-shRNA and i 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 histogram from three independent experiments. Par-4^+/+ and Par-4^−/− MEFs were treated with RSL3 (2 μM) for 3 h. Following the treatment, j 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 histogram from three independent experiments. k U87MG and l 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 histogram 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.