Extended Data Fig. 7: MISO is involved in the recruitment of mitochondrial nucleoids and modulates the level and activity of OXPHOS complexes.

a. Representative images of co-staining for the Mito–GFP and mtDNA/mtRNA (labelled via FASTKD2). Scale bars: main panels 10 μm, magnified insets 2 μm. b. Representative images showing the spatial association between mtDNA and MISO-enriched subdomains in U2OS cells with or without EB treatment. Scale bars: main panels 10 μm, magnified insets 2 μm. c. Quantification of DNA enrichment in subdomains from (b). n = 21, 21cells for Vehicle and EB (3 d), respectively. Three biological replicates. d. Representative expansion microscopy images showing the association between MISO and TFAM in U2OS cells. Scale bars: main panel 20 μm (post-expansion), magnified insets 5 μm (post-expansion). e. Representative expansion microscopy images depicting distinct types of mitochondrial subdomains associated with TFAM in U2OS cells. Scale bars: 5 μm (post-expansion). f. Representative time-lapse images of mtDNA (labelled with PicoGreen) and PHB1-mCherry (marking MISO) in U2OS cells. Arrowheads indicate the subdomain. Scale bar: 2 μm. 1g. Representative images showing the spatial association among MISO-enriched subdomains, mtDNA and replicating DNA (labelled with EdU) in U2OS cells. Scale bars: main panels 10 μm, magnified insets 2 μm. h. Representative images showing the spatial association among MISO-enriched subdomains, mtDNA and SSBP1 in U2OS cells. Scale bars: main panels 10 μm, magnified insets 2 μm. i. Immunoprecipitation with anti-Flag validates the interaction between MISO and SSBP1. j. Representative images of mitochondrial subdomains in U2OS cells expressing MISO–Flag with SSBP1 knockdown. k. Immunoblots showing the knockdown efficiency of SSBP1 in U2OS cells. l. Representative images of U2OS cells expressing PHB2–mNeonGreen and MISO–Flag incubated with MitoSOX or TMRM. MitoSOX or TMRM intensities were represented by calibrated colour scale. Scale bars, 2 μm. m. The gating strategy for determining TMRM fluorescence intensity. n. Flow analysis and quantification of TMRM fluorescence intensity in WT and MISO-KO U2OS cells. n = three biological replicates. o. Seahorse analysis of the mitochondrial respiratory capacity in WT and MISO-KO U2OS cells. n = six biological replicates. p. Flow cytometry analysis and quantification of TMRM fluorescence intensity in U2OS cells expressing an empty vector (pLenti) or MISO–Flag. n = three biological replicates. q. Seahorse analysis of the mitochondrial respiratory capacity in U2OS cells expressing an empty vector (pLenti) or MISO–Flag. r-s. Immunoblots (r) and corresponding quantifications (s) of indicated proteins in MISO-WT, MISO-KO and MISO-OE U2OS cells. n = three biological replicates. t-u. Immunoblots (t) and corresponding quantifications (u) of mitochondrial OXPHOS complexes in liver tissues from mMISO^flox/flox and mMISO^−/− mice. Each lane represents an individual mouse. n = four biological replicates. v-w. Blue-native PAGE-based in-gel activity assays (v) and corresponding quantification (w) of mitochondrial respiratory complexes I, II, IV, and V in WT and MISO-KO U2OS cells. n = three biological replicates. Data are presented as mean ± SD. (c): two-tailed non-parametric Mann-Whitney U-test; (n), (o), (p), (q), (u) and (w): two-tailed unpaired Student’s t-test; (s): ordinary one-way ANOVA with Tukey’s multiple comparisons test.