The mtRNAPol- and mtTFB2-knockdown display reduced mtDNA content and increase the level of unbound TFAM. (A) Immunofluorescence staining of the IFM of TFAM-Apex2 GAL4 control, TFAM-Apex2 under mtRNAPol-, mtTFB1-, and mtTFB2-RNAi flies. Red: TFAM-Apex2; green: dsDNA. (B) The analyses of immunofluorescence intensities of mtDNA and TFAM-Apex2 in TFAM-Apex2 GAL4 control and TFAM-Apex2 in mtRNAPol-, mtTFB1-, and mtTFB2-RNAi flies were plotted in box-and-whisker graphs. (C) The analyses of immunofluorescence colocalization of mtDNA with TFAM-Apex2 and TFAM-Apex2 with mtDNA of TFAM-Apex2 GAL4 control and TFAM-Apex2 in mtRNAPol-, mtTFB1-, and mtTFB2-RNAi flies were plotted in box-and-whisker graphs. The t-test was performed and statistically significant differences between indicated groups are marked with asterisks (n=9–12 volumes of 84.2×84.2×5 μm3 were analyzed, *P<0.05; **P<0.01, compared to the TFAM-Apex2 GAL4 control).