Fig 2: Effects of the C-terminal mTFAM truncations. (A) 26 C-terminal aa residues of mTFAM are dispensable for mtDNA replication. Genotyping as per diagrams in Figure 1C,D using primers listed in Table S1. B, C, C-terminal truncations did not result in a significant reduction in mtCN. A time course of mtCN over 6 days. (B) Most error bars are smaller than the symbols. Representative results of two independent experiments. (C) Cumulative mtCN data over the four time points in B. One-way ANOVA with post hoc Tukey’s test. ns, not significant. (D) Expression of truncated mTFAM variants and MT-CO1 in GeneSwapped cells. A representative of two independent experiments. Calculated molecular weights of polypeptides are presented in kilodaltons. (E) % ATP derived from OXPHOS in GeneSwapped cells with various mTFAM variants. Representative results from two independent experiments with at least triplicate measurements per experimental condition. One-way ANOVA with post hoc Tukey’s test. *, p < 0.05; ****, p < 0.0001. (F) Growth of GeneSwapped cells expressing various mTFAM variants in either +UP or -UP media. A representative of two biological experiments each with three technical replicas. Note that cells expressing the mTFAMΔ27 variant fail to grow in -UP medium, which is consistent with their ρ0 status. (G) Steady-state levels of the MT-RNR2 transcript (HSP1 promoter) are affected the most by TFAM truncations. Cumulative results of at least four biological experiments, each with at least two technical replicates. Mean ± SD. One-way ANOVA with Post hoc Tukey’s test. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Fig 3: TFAM inactivation in 3315#2 murine fibroblasts. (A) mTFAM inactivation is accompanied by mitochondrial expression of EYFP because of the simultaneous excision of the terminator cassette preceding the mitochondrially targeted EYFP gene in the ROSA26 locus. (B) TFAM inactivation is accompanied by the loss of mtDNA. The 3315#2 cells were transduced with adenovirus encoding Cre recombinase (Ad5CMVCre), and EYFP-positive clones were collected via FACS and plated in +UP medium. The resulting colonies were genotyped via PCR for the presence of mtDNA and deletion of TFAM exons 6 + 7 using the primers listed in Table S1. (C,D) TFAM deletion is accompanied by the loss of viability in -UP medium. Cells were seeded at 1 × 104 (10K), 2 × 104 (20K), or 3 × 105 (300K) per well of the 6-well plate, allowed to attach overnight, photographed, and counted (3 wells per cell density). The remaining wells received either +UP or -UP media and were incubated for an additional 96 h, after which photographing and counting were repeated. Representative results of two independent experiments, with triplicate measurements per experimental condition. Means ± SDs.