Fig. 4: mtDNA is conveyed to the cytosol by MDVs.

For all panels: n = 3 independent experiments (unless otherwise specified). In b,c,f,h,i, cFh1^fl/fl cells were treated with 400 μM MMF or vehicle (DMSO) for 6 days. For immunofluorescence experiments, mitochondria or MDVs were labelled using anti-TOM20, anti-PDH and anti-TFAM antibodies and DNA with an anti-DNA antibody. a, TEM images of Fh1^+/+ and Fh1^−/− mouse kidney showing potential vesicle budding at the mitochondrial surface (black arrowheads). Scale bars, 1 μm. b, Representative confocal images of cFh1^fl/fl cells transfected with the indicated siRNAs. Red arrowheads: TOM20⁻PDH⁺DNA⁺ MDVs; blue arrowheads: TOM20⁻PDH⁺DNA⁻ MDVs; white arrowheads: cytosolic DNA foci; green arrowheads: stalled TOM20⁻PDH⁺DNA⁺ MDV budding events. Scale bar, 10 μm. c, Representative N-structured illumination microscopy (N-SIM) super-resolution images of MMF-treated cFh1^fl/fl cells. Red arrowheads: TOM20⁻PDH⁺DNA⁺ MDV budding event; blue arrowheads: TOM20⁻PDH⁺DNA⁺-released MDVs. Scale bar, 5 μm. d,e, Quantification of TOM20⁻PDH⁺DNA⁺ MDVs (d), and compared to cytosolic DNA foci (e) in cFh1^fl/fl cells; P values are relative to the corresponding DMSO-treated control. f, Representative confocal images of MMF-treated cFh1^fl/fl cells. White arrowheads: TOM20⁻PDH⁺TFAM⁺ MDVs. Scale bar, 10 μm. Bottom left, histogram showing the quantification of TOM20⁻PDH⁺ MDVs that are positive for TFAM. g, Quantification of mtDNA copy number by ddPCR using a mtCo3 probe from isolated cytosolic fractions of MMF-treated cFh1^fl/fl cells (12 h or 24 h), and pre-transfected with the indicated siRNAs. n = 4 independent experiments. h,i, Quantification of TOM20⁻PDH⁺DNA⁺ MDVs (h) and compared to cytosolic DNA foci (i) in MMF-treated cFh1^fl/fl cells and pre-transfected with the indicated siRNAs. j,k, Immunoblots of specified proteins (j) and Ifnb1 mRNA expression measured by qRT–PCR (k) in cFh1^fl/fl cells transfected with the indicated siRNAs and treated with MMF for the indicated period of time. Data are mean ± s.e.m. d,g–i,k, One-way ANOVA with Tukey’s multiple comparisons test; e, two-way ANOVA with Tukey’s multiple comparisons test.

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