FAM43A is a cytoplasmic protein under basal and mtDNA-depleted conditions. (A) Representative Airyscan imaging of FAM43A (green) and HSP60 (mitochondria; magenta) in untreated (UNT) and ddC-treated IMR90 cells (20 µM; 96 h). (B) Quantification of Manders overlap coefficient of FAM43A vs. HSP60 and replicating mtDNA vs. HSP60 (positive control of colocalization). (C) Representative Airyscan imaging of FAM43A (green) and LAMP1 (lysosomes; magenta) in UNT and ddC-treated cells. (D) Quantification of Manders overlap coefficient of FAM43A vs. LAMP1. (E) Representative Airyscan imaging of FAM43A (green) and LC3 (autophagosomes: magenta) in UNT and ddC-treated cells. (F) Quantification of Manders overlap coefficient of FAM43A versus LC3. (G) Representative Airyscan imaging of FAM43A (green) and Giantin (Golgi: magenta) in UNT and ddC-treated cells. (H) Quantification of Manders overlap coefficient of FAM43A versus Giantin. In A, C, E, and G, the indicated regions are enlarged on the right with the merge of the two signals shown in the lower right corner. Cells were stained with Hoechst to visualize Nuclei (blue) where indicated. n = 7–11 cells/condition from two independent experiments. Scale bar: 10 µm. Error bars represent mean ± SEM, Unpaired Student’s t test; ns, P > 0.05; ****, P < 0.0001.