FAM43A is an early responder to mtDNA depletion. (A) Heatmap representing the mean log₂ fold-change values of differentially expressed genes in IMR90 cells treated with ddC (20 µM) for the indicated time (n = 2–3 biological replicates per condition) relative to untreated (UNT) control cells. The heatmap shows the expression of the 151 significantly up- and downregulated genes identified in IMR90 cells treated with ddC for 96 h and their expression across all time points. (B) Log₂ fold-change values of GDF15 and FAM43A between UNT and IMR90 cells treated with ddC for the indicated times. Data points represent the mean log₂ fold-change from three biological replicates. Error bars represent mean ± SD. (C) Relative FAM43A mRNA levels in IMR90 cells treated with ddC for 48 h relative to UNT control cells measured by RT-qPCR normalized to ACTIN (n = 3 technical triplicates). Error bars represent mean ± SD, Unpaired Student’s t test; *, P < 0.05. (D) Representative Airyscan images of endogenous FAM43A (green) and nuclei (blue) in UNT cells (top), cells treated with ddC (+ddC) for 48 or 96 h, and cells 96 and 192 h after removal of ddC (Recovery). Insets show the morphological change of FAM43A during mtDNA depletion and repletion. Scale bar: 10 µm. (E and F) Quantification of the fluorescence intensity of FAM43A signal and (F) number of FAM43A aggregates per cell in UNT (n = 10 from two biological replicates). ddC-treated (48 h [n = 9 from two biological replicates] and 96 h [n = 7 from two biological replicates]), and cells 96 (n = 15 from two biological replicates) and 192 h (n = 11 from two biological replicates) after removal of ddC. Error bars represent mean ± SEM, multiple unpaired Student’s t test; ns, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001.