TFAM modulation does not rescue the decreased body weight or the increased spleen/body weight and heart/body weight ratio.

A) Body weight in g. B) Spleen to body weight ratio (g/g). C) Heart to body weight ratio (g/g). D) Testis to body weight ratio (g/g). n≥7. Data are represented as mean ± SEM; *p< 0.05; **p< 0.01; ***p< 0.001; ns: non-significant.

Modulation of TFAM affects mtDNA copy number in a tissue-specific manner.

A) Schematic of the mtDNA highlighting the position of the probes used for mtDNA copy number analysis by qPCR. The deleted mtDNA region is indicated with a grey arc. B-F) Relative mtDNA copy number quantification (Nd1/18S, Atp6/18S, Cytb/18S) in B) liver. C) heart D) colon E) brown adipose tissue and F) spleen. n≥5. Data are represented as mean ± SEM; *p< 0.05; **p< 0.01; ***p< 0.001; ns: non-significant.

Relative TFAM-to-mtDNA ratios in different tissues.

The TFAM-to-mtDNA ratio was calculated from normalized TFAM protein levels (n=2) and normalized mtDNA levels (n=5) as determined by the ND1 probe.

Moderate TFAM overexpression negatively impacts mtDNA gene expression and tissue physiology in liver and correlates with increased FGF21 levels.

A) Relative expression levels of mtDNA encoded transcripts (Nd1/β-actin, Atp6/β-actin, Cytb/β-actin) measured by RT-qPCR in liver. n≥7. Data are represented as mean ± SEM; *p< 0.05; **p< 0.01; ***p< 0.001; ns: non-significant. B) Western blot analysis of steady-state levels of mitochondrial proteins in liver. C) Relative enzyme activities of OXPHOS complexes measured by spectrophotometry in liver mitochondria. n ≥ 6 Data are represented as means ± SEM; *P < 0.05; **P < 0.01; **p< 0.01; ***P < 0.001; ns: non-significant. D) Relative expression levels of mitochondrial stress markers (Atf4/β-actin, Atf5/β-actin, Mthfd2/β-actin) measured by RT-qPCR in liver. n ≥7. Data are represented as means ± SEM; *P < 0.05; **p< 0.01; ***P < 0.001, ns: non-significant E) Quantification of FGF21 levels in plasma measured by ELISA. n ≥ 9.Data are represented as means ± SEM; *P < 0.05; **p< 0.01; ***P < 0.001; ns: non-significant.

Alteration of TFAM does not affect the heart phenotype of mtDNA mutator mice.

A) Relative expression levels of mtDNA encoded transcripts (Nd1/β-actin, Atp6/β-actin, Cytb/β-actin) measured by RT-qPCR in heart. n ≥ 7. Data are represented as mean ± SEM; *p< 0.05; **p< 0.01; ***p< 0.001; ns: non-significant. B) Western blot analysis of steady-state levels of mitochondrial proteins in heart. C) Relative expression levels of mitochondrial stress markers (Atf4/β-actin, Atf5/β-actin, Mthfd2/β-actin, Anf/β-actin) measured by RT-qPCR in heart. n ≥ 7. Data are represented as mean ± SEM; *p< 0.05; **p< 0.01; ***p< 0.001; ns: non-significant.

Increased TFAM levels do not rescue the reduced OXPHOS function in colon of mtDNA mutator mice.

A) Relative expression levels of mtDNA encoded transcripts (Nd1/β-actin, Atp6/β-actin, Cytb/β-actin) measured by RT-qPCR in colon. n ≥7. Data are represented as mean ± SEM; *p< 0.05; **p< 0.01; ***p< 0.001; ns: non-significant. B) Western blot analysis of steady-state levels of mitochondrial proteins in colon. C) BN-PAGE and in-gel activities of complex I and complex IV activities in mitochondrial protein extracts from mouse colon. Coomassie staining of the gel is shown to indicate equal loading. SC, Supercomplexes. D) Relative enzyme activities of OXPHOS complexes measured by spectrophotometry in colon mitochondria. n ≥3. Data are represented as mean ± SEM; *p< 0.05; **p< 0.01; ***p< 0.001; ns: non-significant. E) Relative expression levels of mitochondrial stress markers (Atf4/β-actin, Atf5/β-actin, Mthfd2/β-actin) measured by RT-qPCR in colon. n ≥9. Data are represented as mean ± SEM; *p< 0.05; **p< 0.01; ***p< 0.001; ns: non-significant.

Reduction of TFAM levels in brown adipose tissue has beneficial effects.

A) Relative expression levels of mtDNA encoded transcripts (Nd1/β-actin, Atp6/β-actin, Cytb/β-actin) measured by RT-qPCR in brown adipose tissue (BAT). n ≥5. Data are represented as mean ± SEM; *p< 0.05; **p< 0.01; ***p< 0.001; ns: non-significant. B) Western blot analysis of steady-state levels of UCP1 and mitochondrial proteins in BAT. C) Relative expression levels of brown adipose stress markers (Ucp1/β-actin, Cidea/β-actin, Dio2/β-actin) measured by RT-qPCR in BAT. n≥5. Data are represented as mean ± SEM; *p< 0.05; **p< 0.01; ***p< 0.001; ns: non-significant.

TFAM overexpression restores IL-5 and CCL2 cytokine levels in spleen of mtDNA mutator mice.

A) Relative expression levels of mtDNA encoded transcripts (Nd1/β-actin, Atp6/β-actin, Cytb/β-actin) measured by RT-qPCR in spleen. n ≥5. Data are represented as mean ± SEM; *p< 0.05; **p< 0.01; ***p< 0.001; ns: non-significant. B) Western blot analysis of steady-state levels of mitochondrial proteins in spleen. C) Quantification of IL-5 and CCL2 cytokine levels in plasma measured by the Mouse Cytokine/Chemokine 44-Plex Discovery Assay. n ≥6. Data are represented as mean ± SEM; *p< 0.05; **p< 0.01; ***p< 0.001; ns: non-significant. D) Quantification of Il-5 and Ccl2 cytokine transcript levels in spleen measured by RT-qPCR. n ≥10. Data are represented as mean ± SEM; *p< 0.05; **p< 0.01; ***p< 0.001; ns: non-significant. E) Relative expression levels of immune cell markers (Tbx21/β-actin, Gata3/β-actin, Rorc/β-actin, Foxp3/β-actin, Cebpe/β-actin) for analyzing immune cell populations measured by RT-qPCR in spleen. n ≥5. Data are represented as mean ± SEM; *p< 0.05; **p< 0.01; ***p< 0.001; ns: non-significant. F) H&E staining of spleen sections. scale bar:500µm.

Mating strategy to generate mtDNA mutator mice in combination with the Tfam+/OE or Tfam+/- alleles.

Quantification of mtDNA levels by Southern blot analysis.

A) A. Southern blot analysis of SacI-digested DNA derived from the liver. mtDNA was quantified by radiolabeling with a specific probe against Cytb, nuclear DNA was probed with a specific probe against 18S rDNA. B) Relative Southern blot quantification (mtDNA/18S rDNA).

Cytokine quantification in plasma measured by the Mouse Cytokine/Chemokine 44-Plex Discovery Assay.

A) Interferon Gamma (IFNγ). B) Interleukin 3 (IL-3). C) Tumor Necrosis Factor Alpha (TNFα). D) Macrophage Inflammatory Protein-1 Alpha (MIP-1α). E) Interleukin 2 (IL-2). F) Interleukin 12p70 (IL-12p70). G) C-X-C Motif Chemokine Ligand 1 (KC). H) Interleukin 13 (IL-13). n ≥6. Data are represented as mean ± SEM; *p< 0.05; **p< 0.01; ***p< 0.001; ns: non-significant.

Differential expression analysis of quantitative proteomic data from the spleen.

A) Heatmap of proteins involved in the heme biosynthesis pathway. B) Heatmap of proteins involved in ROS defense. Color indicates the z-score.