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. 2022 Jul 11;11(14):2168. doi: 10.3390/cells11142168

Figure 3.

Figure 3

Coelacanth TFAM promotes spontaneous loss of hmtDNA. (A), Upon cultivation in +UP media, mtCN in cells expressing coelTFAM is reduced. Cells expressing coelTFAM were incubated in +UP media for up to 35 days, cell aliquots were collected at indicated intervals, and mtCN was determined by qPCR. (B), coelTFAM promotes spontaneous loss of mtDNA. Cells were grown in +UP media for 5 weeks, plated by limiting dilution, the resulting colonies were picked and tested for the presence of mtDNA. Red vertical arrows indicate clones that lost mtDNA. (C,D), in the experiment described in (B), clones representative of various mtCNs were analyzed by conventional (C) and dddPCR. (D). (E), Transduction with hTFAM augments mtCN in cells expressing coelTFAM. Cells expressing coelTFAM were selected in –UP media and transduced with a retrovirus rv.5132 (Supplementary Figure S2), which expresses hTFAM. ****, p < 0.0001. (E), One-way ANOVA with post hoc Tukey test. A representative of two independent experiments. (F–H), Changes in OXPHOS and mtDNA-encoded OXPHOS subunits expression correlate with expression of the MT-RNR2. (F), OXPHOS deficiency in cells expressing coelTFAM can be partially rescued with hTFAM. ****, p < 0.0001. One-way ANOVA with post hoc Tukey test. A representative of two independent experiments. (G), coelTFAM does not significantly affect expression of the MT-ND1, MT-CO1, or MT-ND6, but affects expression of the MT-RNR2. Two-way ANOVA with Dunnet correction. ****, p < 0.0001; ns, not significant. (H), OXPHOS correlates with expression of mtDNA-encoded subunits MT-CO1 and MT-CO2. A representative of two independent experiments.