Figure 3.

Mitochondrial Localization of PB1-F2 Protein Is Required for Oxidized DNA Release

(A and B) HEK293FT cells were transfected with siRNA targeting Tom40, Tom22, or control siRNA. Two days later, cells were transfected with the expression plasmid encoding EGFP or PB1-F2 protein in the presence of poly(I:C). Cell lysates were collected at 24 h post transfection and blotted using the indicated antibodies (A). Cytosolic mtDNA was assessed by quantitative PCR at 24 h post transfection (B).

(C) HeLa cells were transfected with expression plasmids encoding PB1-F2 or its C-terminal truncated mutant. At 24 h post transfection, cells were stained with anti-PB1-F2 and anti-Tom20 antibodies to visualize the PB1-F2 protein and mitochondria, respectively, and analyzed by confocal microscopy. Scale bars, 10 μm.

(D and E) HEK293FT cells were transfected with the expression plasmid encoding EGFP, PB1-F2, or its C-terminal truncated mutant in the presence of poly(I:C). Pure cytosolic extracts were collected at 24 h post transfection. Cytosolic mtDNA was assessed by quantitative PCR (D). Oxidized DNA in the cytosol was detected by dot blot analysis using anti-8OH-dG antibody (E). These data are from three independent experiments (B, E; mean ± SEM).

∗∗∗p < 0.001 (one-way ANOVA and Tukey's test).