Fig 2: Immunoreactivity patterns of the mtDNA binding transcription factor A (Tfam) are differentially modified following exposures to cisplatin and doxorubicin. (A‐C) Representative images and quantitation of staining intensity of nontreated and drug‐exposed B16F10 cells show changes in Tfam intensity and distribution, with discrete aggregated pattern following cisplatin and doxorubicin exposures. Reduced staining intensity is seen following hypoxia. Hypoxia lessens drug‐induced Tfam aggregation when compared to normoxic conditions. (D–F) Representative images and quantitation of Tfam immunoreactivity in control and drug‐exposed YUMM1.7 cells. Staining patterns are differentially modified by cisplatin and doxorubicin with aggregation and spindle‐shaped cell morphology. Hypoxia attenuates the steep decline in staining intensity observed after normoxic drug exposures with reduced aggregation and fewer spindle‐shaped stressed cells present. Tfam is observed in brown, counterstained with hematoxylin, and captured with 60x oil objective; scale bar = 20 µm. Staining intensity quantification is shown in panels B and E; bars represent intensity values of TFAM immunoreactivity (a.u.). Asterisk indicates different from nonexposed normoxic or hypoxic control, respectively. In panels C and F, intensity values are normalized and presented as percent change relatively to each respective control; asterisk indicates different from the change measured for each drug exposure under normoxic condition. Data are graphed as mean ± SEM of 3 biological replicates per condition, *p < 0.05. Two‐tailed t‐test was used.

Fig 3: Widefield microscopy-based quantification of cytosolic dsDNA+ foci relative to LMNB1+ nuclear structures, COX4+ mitochondrial structures and TFAM(A–D). Wild-type mouse mammary adenocarcinoma TS/A cells were maintained in control conditions or subjected to γ irradiation (8 Gy) and cultured in control conditions for 24 h, then stained with DAPI (blue nuclear counterstain, A–D) plus dsDNA-specific (red, A–D) and LMNB1-specific (green, B), COX4-specific (green, C) or TFAM-specific (green, D) antibodies and processed for widefield microscopy. Representative images and quantitative data are reported. Scale bar = 30 μm. Results are means ± SEM plus individual data points based on 6-18 images from 2 biologically independent samples over 2 independent experiments. p values are reported, as obtained by unpaired t test(A) or Fisher’s exact test, as compared to 50%-50% random distribution (B). See also Table 1.

Fig 4: MMF treatment phenocopies Fh1 loss.a, Expression levels of the transcriptional markers of Fh1 loss Hmox1 and Nqo1 as well as Tfam expression in monomethyl fumarate (MMF)-treated cFh1fl/fl cells for 8 days compared to vehicle (DMSO)-treated cells, measured by qRT-PCR. n = 3 independent experiments. Bar graphs show the fold change expression, for which the expression in control samples was set to 1. b, Relative abundance of fumarate levels measured by LC–MS in iFh1fl/flCL29 untreated (NT) or treated with vehicle (iFh1fl/flCL29) or 4-OHT (iFh1-/-CL29) or 400 µM MMF for the indicated period of time. n = 5 independent experiments. c, Relative abundance of 2SC levels measured by LC–MS in iFh1CL29 cells and iFh1fl/flCL29 treated with 400 µM MMF for the indicated period of time. n = 5 independent experiments. d, Representative confocal images of cFh1fl/fl cells treated with vehicle (DMSO) or 400 µM MMF for the indicated period of time. Mitochondria and succinated proteins were labelled using anti-TOM20 and anti-2SC antibodies, respectively. Blue arrows indicate 2-SC-decorated proteins accumulation in mitochondria. Scale bar: 10 µm. e-g, Classification of mitochondrial morphology (e), quantification of mitochondrial number (f), and area (g) in cFh1fl/fl cells treated with DMSO or 400 µM MMF for 8 days. n = 3 independent experiments. Data are mean ± s.e.m. a–c, one-way ANOVA with Tukey’s multiple comparison test, e, two-way ANOVA with Tukey’s multiple comparison test, f,g, one-way ANOVA with Tukey’s multiple comparison test, for (e) p-values are indicated above each specific phenotype and relative to the corresponding phenotype in vehicle (DMSO)-treated control. Source data

Fig 5: mtDNA is conveyed to the cytosol by MDVs.For all panels: n = 3 independent experiments (unless otherwise specified). In b,c,f,h,i, cFh1fl/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 cFh1fl/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 cFh1fl/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 cFh1fl/fl cells; P values are relative to the corresponding DMSO-treated control. f, Representative confocal images of MMF-treated cFh1fl/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 cFh1fl/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 cFh1fl/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 cFh1fl/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.Source data