Extended Data Fig. 9: IMM stress stabilizes MISO to promote SMEM formation.

a. CHX chase assay assessing MISO protein stability in U2OS cells treated in the presence or absence of EB. b. Quantification of indicated protein levels from (a). n = three biological replicates. c. Representative images of the Drosophila larval fat body harbouring GFP-marked somatic clones of the indicated genotypes, immunostained for HA-tagged endogenous dMISO. Scale bars: 50 μm. d. Quantification of relative MISO fluorescence intensity from (c). n = 20, 25, 22, 23, 25, 20, 20, 20, 21, 23, 28 cell clones for Luc-i, NP15.6-i, ND-49-i, ND-23-i, SdhC-i, UQCR-Q-i, RFeSP-i, COX6C-i, COX5A-i, ATPsyn-beta-i-1 and ATPsyn-beta-i-2, respectively. Three biological replicates. e. Representative confocal images of the Drosophila larval fat body harbouring GFP-marked somatic clones of dMISO knockdown. Scale bars: 50 μm. f. Quantification of relative MISO fluorescence intensity from (e). n = 20, 20 cell clones for Luc-i and dMISO-i from three biological replicates. g-h. Immunoblots (g) and corresponding quantifications (h) of MISO protein levels in U2OS cells treated for 24 h with the indicated compounds (1 µM Rotenone, 5 mM NaN₃, 2 µM Oligomycin and 2 µM CCCP), except for EB, which was applied at 50 ng/mL for 3 days. Relatively low dosages of the compounds were used to avoid triggering significant mitophagy. n = three biological replicates. i-j. Representative images (i) and corresponding quantifications (j) of PHB2-enriched microdomains (white arrows) in WT and MISO-KO U2OS cells treated with the indicated compounds as described in (g-h). Scale bars: main panels 10 μm, magnified insets 5 μm. n = three biological replicates. k-l. Representative images (k) and corresponding quantification (l) of mitochondrial phenotype in WT and MISO-KO U2OS cells expressing mCherry-Parkin, treated with DMSO or 10 μM CCCP for 12 h. Scale bars: main panels 20 μm, magnified insets 5 μm. n = three biological replicates. m-n. Representative images (m) and corresponding quantifications (n) of PHB2-enriched microdomains (white arrows) in WT and MISO-KO U2OS cells treated with indicated shRNAs. Scale bars: main panels 10 μm, magnified insets 5 μm. n = three biological replicates. o. Immunoblots showing the knockdown efficiency of MIC60 and ATP5A in U2OS cells. p. CHX chase assay assessing MISO protein stability in U2OS cells stably expressing MISO and treated with the indicated shRNAs. q. Quantification of indicated protein levels from (p). n = three biological replicates. r. qPCR analysis of MISO mRNA levels in U2OS cells transduced with the indicated shRNAs. n = three biological replicates. Data are presented as mean ± SD. (b) and (f): two-tailed unpaired Student’s t-test; (d): ordinary one-way ANOVA with Dunnett’s multiple comparisons; (h), (j), (n), (q) and (r): ordinary one-way ANOVA with Tukey’s multiple comparisons test; (l): two-way ANOVA with Tukey’s multiple comparisons test.