Fig 1: Altered mitochondrial protein homeostasis in PISD patient fibroblasts.(A) Western blot analysis of various mitochondrial proteins in control and patient fibroblasts treated with 2-deoxyglucose (20 mM) and 50 μM lyso-PE for 48 h as indicated. VDAC was used as a marker for total mitochondrial signal, whereas β-ACTIN was used as a general load control. Open and solid triangles indicate unprocessed and processed forms, respectively. Decreased levels of OMA1, OPA1, MRPL32, and processed PGAM5 were observed in patient fibroblast cells, but rescued upon lyso-PE treatment. To obtain optimal separation of OPA1 bands, separate gels were run and blotted with HSP60 as a load control. (B) Protein extracts from HEK cells overexpressing wild-type or mutant PISD constructs (as in Fig 8) were analyzed by Western blot for the same proteins as in part (A). Overexpression of wild-type PISD leads to a dramatic decreased in OMA1 and MRPL32, which is blunted when mutant PISD proteins are overexpressed. Similar results were replicated in three independent experiments.
Fig 2: Relative mRNA quantification of OMA1, PGMA5, OPA1, and MRPL32 in control and patient fibroblasts, as well as in HEK cells overexpressing PISD.(A) Relative mRNA levels of OMA1 and OPA1 were similar between control and patient fibroblast. Patient fibroblast mRNA levels of PGAM5 and MRPL32 decreased; however, these patterns were not correlating to the Western blot analysis. (B) Relative levels of OMA1, PGAM5, OPA1, and MRPL32 mRNA are similar across all treatments. Error bars represent SD.
Fig 3: Activation of KEAP1-PGAM5-AIFM1 signaling axis in SNG-induced oxeiptosis.A HT-29 cells were treated with SNG for indicated time and Western blot analysis was carried out. The signal intensities of western blot bands were normalized to actin of each group, and fold changes were plotted in a histogram from three independent experiments. Significant difference, *p < 0.05, **p < 0.01, ***p < 0.001 and ns = no significance. B HT-29 and CaCo-2 cells were treated with SNG for 16 h and 6 h, respectively. Following the treatment, Western blot analysis of indicated proteins was performed. The signal intensities of western blot bands were normalized to AIFM1 of each group, and fold changes were plotted in a histogram from three independent experiments. Significant difference, ***p < 0.001. C Cells were treated with SNG in the presence or absence of NAC, and Western blot analysis of indicated proteins was performed. The signal intensities of western blot bands were normalized to AIFM1 of each group, and fold changes were plotted in a histogram from three independent experiments. Significant difference, **p < 0.01 and ***p < 0.001. KEAP1-shRNAs-transfected HT-29 cells were treated with the indicated concentrations of SNG for 16 h. Following the treatment, D cell viability was performed by MTT assay. Data shown are mean ± SD (n = 3) (***p < 0.001), E crystal violet staining, Scale bar: 10 µm, and F Western blot analysis of indicated proteins was performed. The signal intensities of western blot bands were normalized to AIFM1 of each group, and fold changes were plotted in a histogram from three independent experiments. Significant difference, ***p < 0.001.
Fig 4: Pivotal role of KEAP1-PGAM5-AIFM1 signaling axis in SNG-induced oxeiptosis.CRC cells were stably transfected with PGAM5-shRNAs. After transfection, cells were treated with SNG (4 μM) for 16 h. Following the treatment, A Western blot analysis of indicated proteins was performed. The signal intensities of western blot bands were normalized to AIFM1 or actin of each group, and fold changes were plotted in a histogram from three independent experiments. Significant difference, **p < 0.01 and ***p < 0.001. B CRC cells were stably transfected with PGAM5-shRNAs. After transfection, cells were treated with the indicated concentration of SNG for 16 h and then cell viability was measured by MTT assay. Data shown are mean ± SD (n = 3) (*p < 0.05, **p < 0.01 and ***p < 0.001) and C crystal violet staining was performed. Scale bar: 10 µm. Indicated cells were stably transfected with AIFM1-shRNA, after transfection, cells were treated with the indicated concentrations of SNG (HT-29 for 16 h and CaCo-2 for 6 h), respectively. Following the treatment, D and E cell viability was measured by MTT assay. Data shown are mean ± SD (n = 3) (*p < 0.05, **p < 0.01 and ***p < 0.001), and F crystal violet staining was performed. Scale bar: 10 µm. G HT-29 cells were treated with SNG in the presence or absence of NAC for 16 h. Whole cell lysates of treated as well as untreated cells were subjected to immunoprecipitation with the respective antibodies as indicated, followed by detection precipitates (top) and input lysates (bottom) with the appropriate antibodies by Western blotting. HT-29 cells co-treated with H2O2 (250 μM) were exposed to SNG (2 μM) in the presence or absence of NAC. H Western blot analysis of indicated proteins was performed. The signal intensities of western blot bands were normalized to AIFM1 of each group, and fold changes were plotted in a histogram from three independent experiments. Significant difference, *p < 0.05, ***p < 0.001 and ns = no significance, I Cell viability was measured by using MTT assay. Data shown are mean ± SD (n = 3) (*p < 0.05, ***p < 0.001).
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