Fig 1: CRABP2 promoted chemoresistance by regulating ubiquitination degradation of Bax.a After adding MG-132 to AGS cells with knockdown or overexpression of CRABP2, Co-IP of Bax was performed, and ubiquitin was detected by western blotting. b GC cells with CRABP2 knockdown or overexpression were transfected with HA-labeled ubiquitinated plasmids of different sites, and Co-IP experiments of HA were performed. After adding OXA to AGS/HGC-27 cells, the activities of caspase 9 (c) and caspase 12 (d) were detected. e After adding OXA to AGS/HGC-27 cells, the expression of CC3 was detected. f After adding OXA to AGS/HGC-27 cells, the expression of DNA damage/repair-related proteins was examined by western blotting. g After AGS/HGC-27 cells were treated with OXA, the cytoplasm and mitochondria were separated, and the expression of BAX was examined. h The activities of caspase 9 were detected after CRABP2 knockdown/overexpression in AGS/HGC-27 cells. i The expression of CC3 was examined after CRABP2 knockdown/overexpression and OXA added to AGS/HGC-27 cells. j After adding OXA to AGS/HGC-27 cells and knocking down/overexpressing CRABP2, the expression of DNA damage/repair-related proteins was examined by western blotting. k, l After AGS/HGC-27 cells with CRABP2 knockdown or overexpression were treated with OXA, the cytoplasm and mitochondria were separated, and the expression of BAX was examined. (OXA oxaliplatin, CC3 cleaved caspase 3, TOMM40 translocase of outer mitochondrial membrane 40).
Fig 2: High expression of MTCH2 in glioma cells may increase mitochondrial OXPHOs. a, b Western blots and quantifications showing the enriched expression of MTCH2 in glioma cells compared to primary neurons and astrocytes. c qPCR results showing the decreased mRNA level of MTCH2 in A172 cells by siRNA-mediated knockdown (N = 3). d, e Western blots and quantifications showing the decreased protein level of MTCH2 in A172 cells by siRNA-mediated knockdown. Noted that other mitochondrial outer membrane proteins Tom20 and Tom40 were not affected by MTCH2 knockdown. f Immunostaining images showing the mitochondrial localization of MTCH2 in A172 cells. Noted that MTCH2 signals were dramatically decreased by siRNA-mediated knockdown. Scale bar, 10 µm. g Western blots showing the increased mitochondrial respiratory proteins and 4-HNE levels by MTCH2 knockdown in A172 cells
Fig 3: The effects of mito-HIF-1a are independent of its transcriptional activity. A–D. Protease K sensitivity assay of the indicated mitochondrial proteins. Immunoblot analysis of intact mitochondria isolated from CoCl2-treated HeLa cells (A) or DMOG-treated HeLa (B), HUVEC (C), and HK-2 (D) cells. E. Construction of vectors for expression of mutant mito-HIF-1a proteins lacking the NTAD, ID, or CTAD. aa, amino acid. F. Flow cytometric analysis of apoptosis (annexin V/PI) in transfected HeLa cells after exposure to hypoxia for 16 h. Grp75 was probed as a matrix marker; TIMM17A as an inner membrane marker; cytochrome c as an intermembrane space marker; and TOMM70A, TOMM40, TOMM34, and Bcl-XL as outer membrane markers. Data are means ± SEM (n = 5). ns, not significant; **p < 0.01.
Fig 4: A chemical screen for mitochondrial ceramide-binding proteins yields VDAC1 and -2. a Structure of the photoactive and clickable C15-ceramide analog, pacCer. b Mitochondria isolated from HeLa cells were incubated with liposomes containing increasing amounts of pacCer, UV irradiated, and then click reacted with AF647-N3. Samples were processed for SDS-PAGE, subjected to in-gel fluorescence (IGF, red), and stained with Coomassie blue (CB, blue). p33•Cer denotes a prominently photolabeled protein band of ~33 kDa. c Strategy for the identification of p33•Cer. d p33•Cer was purified from pacCer-labeled and TAMRA/biotin click-reacted mitochondria using NeutrAvidin-beads, imaged by IGF, excised from the gel, digested by trypsin, and then identified by LC-MS/MS. Data from two independent experiments revealed that p33•Cer corresponds to VDAC1 and VDAC2. e Specificity of anti-VDAC antibodies was validated by immunoblotting of mitochondria isolated from HeLa cells treated with non-silencing (siNS) or VDAC-targeting siRNAs (siVDAC1, siVDAC2). Note that the anti-VDAC3 antibody cross-reacts with VDAC2. f Mitochondria isolated from siVDAC1/2-treated HeLa cells were photolabeled with pacCer, click-reacted with AF647-N3, and subjected to IGF analysis followed by CB staining. g Fractions obtained during affinity purification of p33•Cer were subjected to SDS-PAGE, transferred on nitrocellulose, analyzed by on-blot-fluorescence (OBF) and probed with antibodies against VDAC1, TOM40, and p60-Mito. h Fractions obtained during affinity purification of p33•Cer were processed as in g and probed with anti-VDAC2 and anti-VDAC3 antibodies. T total mitochondria extract, FT flow-through, W wash, E eluate
Fig 5: MTX2 deficiency induces altered mitochondrial protein composition and increased mitochondrial fission.a Immunoblot analysis of MTX2 and other outer and inner mitochondrial membrane proteins of whole-cell extracts from healthy control (WT) and patients’ (MADM2, MADM3) fibroblasts. Actin or REVERT total proteins were used as loading controls. n = 3 independent experiments for MTX2, TOMM22, SAMM50, CHCHD3 and n = 2 independent experiments for MTX1, VDAC2, TOMM40. b Representative confocal microscopy images showing mitochondria stained using DAPI (blue) and Mitotracker (red) from a control, patient MADM2, and patient MADM3. Boxed regions are enlarged. Scale bar: 10 µm. Data shown are representative of ten independent experiments. c Mitochondrial network analysis using the MINA ImageJ macro of healthy and patients’ fibroblasts showing significant differences in: mitochondrial footprint (exact p values: pMADM2 = 0.026, pMADM3 = 0.041), individuals (pMADM2 = 0.013, pMADM3 = 0.042), networks (pMADM2 = 7.31737E-05, pMADM3 = 0.041) and mean number of branches per network (pMADM2 = 0.005, pMADM3 = 0.043). 53 (WT), 46 (MADM2) and 51 (MADM3) cells from n = 5 independent experiments were blindly scored. Box plots show median (horizontal lines), first to third quartile (box), and the most extreme values within 1.5 times the interquartile range (vertical lines). Two-tailed unpaired t test; *p < 0.05, **p < 0.01, ***p < 0.001. d Immunoblot analysis of OPA1-L (protein optic atrophy 1, long) and OPA1-S (protein optic atrophy 1, short) (proteins involved in mitochondrial fusion, OPA1-S being issued from cleavage of OPA1-L) and DRP1 (protein involved in mitochondrial fission). Protein levels were quantified by ImageJ software and their expression levels were normalized to actin as an internal loading control. Results are expressed as mean ± SD, two-tailed unpaired t test was used to evaluate the statistical significance of differences among the groups (exact p values: OPA1-L: pMADM2 = 0.772, pMADM3 = 0.385; OPA1-S: pMADM2 = 0.10, pMADM3 = 0.10; DRP1: pMADM2 = 0.0015, pMADM3 = 0.042). *p < 0.05, **p < 0.01, ns not significant (n = 3 and n = 4 independent experiments respectively for OPA1 and DRP1). Source data are provided as a Source Data file.
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