Fig. 1: Specific targeting of mitochondrial DNA by the FokI endonuclease reduces the amount of mitochondrial DNA.

a Schematic representation of the domain structure of the mitochondrial DNA-targeting FokI endonuclease. b Western blot analysis to confirm the expression of the indicated mt-FokI in the HeLa cell line harvested 36 h after transfection. WT, wild type, active mt-FokI; MT, catalytically inactive point mutant. c Representative immunofluorescence images visualizing the mitochondrial localization of TFAM-FokI in HeLa cell lines stably expressing mito-Dendra2 fluorescent protein using structured illumination microscopy. Scale bars, 10 μm. d Western blot analysis of mt-FokIs in nuclear, cytosolic, and mitochondrial fractions isolated from HeLa cell line transfected with the indicated mt-FokI. N, nuclear; C, cytosolic; M, mitochondrial fraction. e Immunofluorescence staining with anti-DNA antibody in HeLa cell line transfected with TFAM-FokI. Representative images with expressed TFAM-FokI in red and stained dsDNA in green. Scale bars, 10 μm. f Quantitative PCR (qPCR) for relative mtDNA copy number using primers in the D-loop, MTCOX2 sequence in the HEK293T cell line, 36 h after indicated mt-FokI expression. Normalized expression Data are means ± SD of n = 4. Statistics were performed using one-way analysis of variance (ANOVA) with confidence interval = 95% and Bonferroni’s post hoc test. g Western blot analysis of endogenous TFAM in the same samples as shown in (f). Each experiment was performed at least three times. *P ≤ 0.05, ***P ≤ 0.005, ns, not significant.