Fig. 3. Drp1 phosphorylation is essential for Acsl4-dependent ferroptosis.

A SDS-PAGE silver staining showed typical pull-down results of Acsl4 after incubation with PL1 cell lysate. Mass spectrometry identified the band framed in the oval as Drp1. B The interaction between Acsl4 and Drp1 was confirmed by co-immunoprecipitation in PL1 and PG7 cells. C Confocal images showed colocalization of Acsl4 (red) and Drp1 (green) in PL1 and PG7 cells. Nuclei were counterstained with Hoechst (blue). Scale bars: 10 μm. D, E Expression levels of p-Drp1^Ser637, p-Drp1^Ser616, and Drp1 were determined by western blot in PL1 and PG7 cells in the presence or absence of erastin (5 μM, 6 h). Drp1 was used as a loading control of two types p-Drp1. GAPDH was used as control. Data indicated as mean ± S.D. (n = 4 experiments). F Representative images of IHC staining of p-Drp1^Ser637, p-Drp1^Ser616, and Drp1 in two pairs LGG and GBM tissues. Scale bars: 100 μm. Data indicated as mean ± S.D. (n = 4 experiments). G Flow cytometric analysis of p-Drp1^Ser637 and p-Drp1^Ser616 levels in PL1 and PG7 cells. Isotype control was set in gray. The histogram shows mean fluorescence intensity (MFI) values for control and erastin-treated cells. Data indicated as mean ± S.D. (n = 4 experiments). H Confocal images showed colocalization of oxidized lipids (green) and mitochondria (red). PL1 cells of indicated groups were treated with erastin (1 μM, 6 h), and PG7 cells of the indicated groups were treated with erastin (2 μM, 6 h); then cells were stained with BODIPY C11 and MitoTracker. Scale bar: 10 μm. I–J Intracellular ROS and MDA level in PL1 and PG7 cells treated as indicated before. Data indicated as mean ± S.D. (n = 3 experiments). *p < 0.05, **p < 0.01, ***p < 0.001.