Depletion of mtDNA in IMR90 cells as a model of intergenomic signaling. (A) Relative mtDNA copy number of cells treated with ddC (20 µM) for the indicated time measured by qPCR with ND1 primers, normalized to nuclear B2M. The relative mtDNA copy number of untreated (UNT) cells was given a value of 100%. Data points represent the mean of three technical replicates from three biological replicates. Error bars represent mean ± SD, one-way ANOVA with Dunnett’s multiple comparisons; ****_, P < 0.0001. (B) Representative fluorescent images and quantification of control UNT cells and cells treated with ddC for the indicated times showing colocalization of EdU (green) and Hoechst-stained (blue) nuclei (n = 10 from two biological replicates). Scale bar: 100 µm. Error bars represent mean ± SD. One-way ANOVA with Dunnett’s multiple comparisons; ns, P > 0.05. (C and D) Mitochondrial mass and (D) membrane potential of control UNT cells and cells treated with ddC for the indicated time. Data points represent the mean of three technical replicates from three biological replicates. Error bars represent mean ± SD, multiple Unpaired Student’s t test; ns, P > 0.05. (E and F) ATP and (F) hydrogen peroxide (H₂O₂) measurements of control UNT cells and cells treated with ddC for the indicated time. Data points represent the mean of three technical replicates from three biological replicates. Error bars represent mean ± SD, multiple Unpaired Student’s t test; ns, P > 0.05. (G) Oxygen consumption rate during Seahorse metabolic flux assay of untreated and cells treated with ddC for the indicated time (n = 4 biological replicates). Error bars represent mean ± SD. (H and I) Basal oxygen consumption and (I) Spare respiratory capacity (from G) of control UNT cells and cells treated with ddC for the indicated time (n = 4 biological replicates). Error bars represent mean ± SD, multiple Unpaired Student’s t test; ns, P > 0.05.