Fig 1: GIPC1 transported DECR1 into mitochondria through the actin-based manner.A The identification of the interacting proteins with GIPC1 by Co-IP/MS analysis. B The molecular docking showing the interaction between GIPC1 (cyan) and DECR1 (green). C SPR analysis showing the direct interaction between GIPC1 and DECR1. D Representative Western blot and quantitative analysis of DECR1 protein in NMCMs with 2 µM DOX for 0, 12, 24, 48 h, respectively. n = 3 per group. E Co-IP assay showing the interaction between Flag-GIPC1 and His-DECR1 after treatment with DOX for 24 h in HEK293 cells. n = 3 per group. F The locations of mitochondria (red), GIPC1 (green) and DECR1 (yellow) in NMCMs treated with DOX for 24 h were detected by IF. scale bar = 10 µm. G The interactions between Flag-GIPC1, Flag-GIPC1-mut, His-DECR1 and His-DECR1-mut detected by co-IP assay. n = 3 per group. H The locations of mitochondria (red), GIPC1 (green) and DECR1 (yellow) in H9c2 cells detected by IF. scale bar = 10 µm. n = 3 per group. I Co-IP assay showing the interaction of MYO6, GIPC1 and DECR1 after treatment with DOX in H9c2 cells. n = 3 per group. J The locations of mitochondria (red), DECR1 (yellow) and phalloidin (green) in H9c2 cells after treatment with DOX for 24 h by IF. scale bar = 10 µm. K The schematic diagram showing the interactions of GIPC1, DECR1 and MYO6. Created with figdraw.com. L The interactions between MYO6, Flag-GIPC1, Flag-GIPC1-mut, His-DECR1 and His-DECR1-mut detected by Co-IP assay. n = 3 per group. M, N The locations of mitochondria (red), DECR1 (yellow) and phalloidin (green) in H9c2 cells after transfected with siRNA or overexpression plasmid against GIPC1, and DECR1 plasmid as well as its mutation in the presence or absence of jasplakinolide/cytochalasin by IF. scale bar = 10 µm. n = 3 per group. All quantitative data were expressed as mean ± SEM, and statistical significance was analyzed using t-test, *P < 0.05, **P < 0.01, ***P < 0.001.
Fig 2: DECR1 overexpression reversed GIPC1 deletion-induced ferroptosis.A Representative Western blot and quantification of DECR1 protein expression in NMCMs transfected with the DECR1 overexpressing or mutation plasmid. n = 3 per group. B Representative TEM images showing the ultrastructure of NMCMs. *: mitochondria with disrupted cristae. scale bar = 1 µm. n = 3 per group. C Representative images and quantitative analysis of mitochondrial membrane potential with TMRE staining in H9c2. scale bar = 20 μm. n = 4 per group. D Representative images and quantitative analysis of C11 BODIPY 581/591 staining in H9c2. scale bar = 20 μm. n = 4 per group. E Representative images and quantitative analysis of MitoSOX staining in H9c2. scale bar = 200 μm. n = 3 per group. F–H MDA content (n = 5 per group), 4-HNE level (n = 3 per group) and GSH/GSSG ratio in NMCMs (n = 5 per group). I Representative images and quantitative analysis of cytoplasmic Fe2+ stained with FerroOrange in H9c2. scale bar = 20 μm. n = 4 per group. J Western blots and quantitative analysis of ACSL4, PTGS2, GPX4 and SLC7A11 protein levels in NMCMs. n = 5 per group. K–M The heatmap showing the statistical significance of the different lipid species in the indicated groups. n = 3 per group. All quantitative data were expressed as mean ± SEM, and statistical significance was analyzed using the one-way analysis of variance (ANOVA), *P < 0.05, **P < 0.01, ***P < 0.001.
Supplier Page from Abcam for Recombinant Human DECR1 protein (His tag N-Terminus)