Fig 1: 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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