Fig 2: M1BP KD leads to reduced mitochondrial respiratory complex assembly.a Volcano plots of differential gene expression analyses between Mef2>WT and Mef2 > M1BP RNAi developing DLM. The top volcano plots represent genes encoding structural subunits and bottom plots assembly factors of mitochondria respiratory complexes. Genes are colour-coded accordingly to their complex membership. For statistical analysis, the Wald test with Benjamini–Hochberg-correction was applied. b Blue native polyacrylamide gel of mitochondrial complexes extracted from adult flight muscle mitochondria. Complexes are depicted by a black circle, V1 and V2 refer to the monomer and dimer of complex V, respectively. Quantifications of the intensity of bands, representative of the protein quantity for each complex are represented in the bar chart as a percentage relative to the WT. c Western Blot of ATP5A Complex V subunit on detergent-soluble and insoluble protein fractions from WT and M1BP-depleted adult purified mitochondria. The Ponceau S membrane staining is shown as a loading control. The mean band intensities are represented as histograms for each condition from two independent biological replicates. Protein size is indicated in kDa. d Representative transmission electron micrographs (with an enlarged view on the right, depicted by a white rectangle) of the ImmunoEM experiment using gold-conjugated secondary antibody targeting an anti-Complex V antibody. The gold conjugates were quantified with respect to their location. Data are represented in boxplots (median and quartiles) with whiskers (minimum to maximum), where each image gave rise to a single value of the density of beads for each of the three locations; n = 9 sections; total number of beads= 1048 in the matrix, 169 in the cytosol and 102 in aggregates. The scale bar corresponds to 2 µm. After applying non-parametric Kruskal–Wallis test the pairwise, two-sided Mann–Whitney test was used (p values = 0.0004 ***). Source data are provided as a Source data file.

Fig 3: Reduction in Alb.−/− lipid droplet (LD) is not associated with β-oxidation A Liver gene expression related to β-oxidation. ACADL: acyl-CoA dehydrogenase long chain; ACOX1: Acyl-CoA Oxidase 1; CPT1-α: Carnitine palmitoyltransferase-Iα; CS: Citrate synthase. B Hepatic ACADL protein expression. C) Hepatic ATP5a1 protein expression. ATP5a1: ATP synthase F1 subunit α. EX: exercise, CON: control. Analysis by ANOVA. n = 6 per group. Data are presented as the mean ± S.E.M. mRNA results normalized to 18S and protein results normalized to β-actin. EX different from CON within a genotype, * P < 0.05

Fig 4: Fluorescent esters as counterstains alongside antibody benchmarks. Combining NHS esters with immunofluorescence provided with spatial context for the localisation of the antigens in HeLa cells. (A) 4× EExM images of antibody labels for the cis-golgi marker GM130 (yellow) and the mitochondrial ATP5A1 ATP synthase (magenta) alongside NHS Alexa488 (cyan) applied pre-gelation. (B) 4× EExM images shows examples of antibody labelling for targets at specific compartments alongside ester counterstains (all applied pre-gelation), including (i) the KDEL endoplasmic reticulum retention sequence, alongside NHS AZ488 revealing aspects of the cytoplasmic reticular network and organelles, (ii) the mitochondria ATP synthase subunit ATP5A1 alongside NHS ATTO647N, and (iii) the cis-golgi matrix protein GM130 alongside NHS BODIPY493/503. Scale bars (expansion factor rescaled): (A) 3.75 μm; (Bi) 2.5 μm, (Bii) 2.5 μm, (Biii) 5 μm.