Additional file 1 of Mitochondrial CISD1/Cisd accumulation blocks mitophagy and genetic or pharmacological inhibition rescues neurodegenerative phenotypes in Pink1/parkin models

Additional file 1: Supplementary Figure S1. Characterisation of Cisd in ageing and different tissues. (A) Immunoblot analysis of protein lysates from young (2 days) or old (30 days) whole flies of the indicated genotypes. Samples were homogenised under non-reducing (top panel) and reducing (bottom panel) conditions. Blots were probed for Cisd (CISD2, Proteintech, 13318-1-AP) and Tubulin. (B) Immunoblot of mitochondrial fractions from WT flies, probed for poly-Ub, Cisd (CISD2, Proteintech, 13318-1-AP) and ATP5a. (C) Immunoblot analysis of protein lysates from 2- and 30-day-old adult heads of the indicated genotypes, probed for pUb, Cisd (CISD2, Proteintech, 13318-1-AP) and Tubulin. (D) Relative amount of Cisd monomer and dimer quantified from replicate blots shown in C. (E) Immunoblot of protein lysates from whole flies of WT or Cisd null (−/−) or Cisd overexpression (OE) driven by da-GAL4, probed for Cisd (CISD2, Proteintech, 13318-1-AP) and Tubulin. (F, G) Climbing assay of 2- and 20-day-old WT flies orCisd overexpression driven only in (F) DA neurons (TH-GAL4) or (G) pan-muscle (Mef2-GAL4). (H) Lifespan analysis of flies overexpressing Cisd in muscles as in G versus a WT control genotype. Statistical analysis: (D) unpaired t-test; (F, G) Kruskal-Wallis non-parametric test with Dunn’s post-hoc correction. ** P < 0.01, **** P < 0.0001. Supplementary Figure S2. Quantification of p62, Atg8-II and Cisd protein levels, and Pink1 and parkin mRNA. (A, B) Quantification of immunoblots shown in Fig. 4D. (C) Quantification of Cisd levels from immunoblots shown in Fig. 4I. (D, E) Quantification of Pink1 (D) or parkin (E) transcript levels upon Cisd overexpression (OE) or knockdown (RNAi). Statistical analysis: (A, B, D, E) Welch’s t test, (C) one-way ANOVA with Sidak’s post-hoc correction. * P < 0.05, ** P < 0.01, **** P < 0.0001. Supplementary Figure S3. Characterisation of Cisd loss-of-function on organismal and mitochondrial phenotypes. (A) Immunoblot analysis of protein lysates from whole flies of the indicated genotypes of Cisd loss versus control, probed for Cisd (CISD2, Proteintech, 13318-1-AP) and Tubulin. (B) Climbing assay of 2- and 20-day-old flies of the indicated genotypes. Knockdown is driven by da-GAL4. (C) Lifespan analysis of Cisd loss as in B. (D) Confocal microscopy analysis of mitochondrial morphology, immunostained for ATP5A mitochondrial marker in flight muscle of the indicated genotypes. Knockdowns are driven by da-GAL4. (E) Confocal analysis of adult neurons of the indicated ages, WT control and Cisd RNAi (KK line) animals co-expressing the mitophagy reporter mito-QC (OMM-localised tandem RFP-GFP) with nSyb-GAL4 to highlight mitolysosomes, shown separately and quantified in F. Statistical analysis: (B) Kruskal-Wallis non-parametric test with Dunn’s post-hoc correction. (F) one-way ANOVA with Sidak’s post-hoc correction. * P < 0.05, **** P < 0.0001. Scale bars = 10 μm. (G) Immunoblot analysis of protein lysates from whole flies of the indicated genotypes probed for pUb and Total Ub. Supplementary Figure S4. Characterisation of Cisdand parkin genetic interaction. (A, B) Climbing assay of tissue-specific Cisd knockdown (KD) in either pan-muscles with Mef2-GAL driver (A) or pan-neurons with nSyb-GAL driver (B) alongside respective controls. (C) Viability assay for genetic interactions between the indicated combinations revealing synthetic lethality of Pink1/parkin mutants and CisdOE with da-GAL driver. (D) Confocal microscopy analysis of mitophagy reporter mito-QC in flight muscle from Cisd knockdown driven by Mef2-GAL4 in WT and parkin mutant backgrounds in 30-day-old animals. (E) Quantification of the number of mitolysosomes shown in D. Data points indicated individual animals analysed. Statistical analysis: one-way ANOVA with Sidak’s post-hoc correction; * P < 0.05, **** P < 0.0001. Scale bars = 10 μm.