NEURL4 is required for the regulation of mtDNA integrity. (A and B) ChIP analysis showing recruitment of NEURL4 at mtDNA sites (A) and decreased levels of poly-ADP-ribosylation associated with mtDNA in N4-KO cells (B). Plots include representative results obtained in three distinct biological replicate experiments, with bar graphs representing the mean ± SD between technical triplicates of one single experiment. (C) No significant change in mtDNA copy number as measured by PCR ratio between mtDNA and nuclear DNA. Data represent the mean ± SD of three experiments. (D) Long-range PCR assay with Cytb divergent primers showing increased mtDNA deletions in N4-KO cells as compared with the HeLa parental line (WT). (E and F) IP assay showing suppression of mtLIG3 PARylation in the absence of NEURL4 (E) or in the presence of the generic PARP inhibitor BGP-15 (F). (G) Quantitative PCR assay showing increased mtDNA accumulation in the cytoplasm of N4-KO cells as compared with parental Hela cells. Data represent the mean ± SD of three experiments. (H) WB showing activation of caspase 1 and IL-1β in N4-KO cells. (I) Long-range PCR assay showing increased mtDNA deletions in spermatocytes from NEURL4^+/− mice (N4-het) compared with WT littermates. (J) Decreased sperm count in NEURL4 heterozygous mice compared with WT littermates. Mean of sperm count across five random fields for four mice/genotype ± SD. Statistical significance calculated by two-tailed Student’s t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Mito, mitochondrial extracts; tot/cyt, ratio of amplification in total and cytosolic extracts; Cytb, Cytochrome b.