Extended Data Fig. 6: Characterisation of MOF variants.
From: COX17 acetylation via MOF–KANSL complex promotes mitochondrial integrity and function
a, Mitochondria from control (iWT) and Mof iKO MEF cells were lysed with digitonin. Protein complexes were subjected to blue native electrophoresis and in-gel activity stain for complex IV. HSP60 immunodetection served as a loading control. b, Immunoblot of cell extracts of control and Mof iKO MEFs to verify the corresponding knockout (KO) efficiency by 2 days of 4-OHT induction. Actin and GAPDH served as loading controls. c, Experimental scheme for subcellular fractionation in MEFs. d, Immunoblot analysis of cell lysate as input and Percoll gradient purified mitochondria for MOF and KANSL3 detection. Quality of fractionation was verified using mitochondrial marker (cytochrome c), nuclear marker (H3) and cytosolic marker (actin). e, Immunoblot for detection of enrichment of MOF in the mitochondria. Cell lysate served as the input for crude mitochondrial fraction. Quality of enrichment was verified using H4K16ac as a nuclear marker. Signal specificity of MOF was verified using Mof iKO cell lysate. f, Structured Illumination immunofluorescence of C-terminal HA-3xFLAG-tagged KANSL2 expressing from FRT locus in HeLa Flp-In T-Rex cells stained with Mitotracker and FLAG antibody. Scale bars: 5 µm. g, h, Immunoblot (g) and immunofluorescence of FLAG-tagged MOF for expression of indicated MOF variants in MEFs. Beta-actin served as a loading control. Insets of mitochondria indicate selected regions of interest. White arrowheads indicate micronuclei. Scale bars: 10 µm. i, Flow cytometry of mtSOX dye in control MEFs expressing the indicated MOF variants (mean ± s.e.m., normalized to the empty vector (EV) control for each experimental replicate, n = 2-6 embryos). j, Flow cytometry of mtSOX dye in control and Mof iKO MEFs expressing indicated MOF variants (mean ± s.e.m., normalized to the corresponding iWT control for each experimental replicate, n = 2-3 embryos, two-tailed Student’s t-test). k, Luminiscent analysis of reduced (GSH) and oxidized (GSSG) glutathione as a measure of anti-oxidant metabolism in control and Mof iKO MEFs. Data is represented as a ratio of GSH:GSSH (mean ± s.e.m. n = 5-6 embryos, two-tailed Student’s t-test). l, anti-FLAG antibody immunoblot of cell lysates of indicated KANSL2 variants in MEFs. GRP75 served as a loading control.
