Figure 8.

AIF translocation induced by PARP-1 or NADase is prevented by pyruvate. A, Immunostaining for AIF (green) in cultures fixed 3 h after incubation with MNNG alone (75 μm for 30 min), MNNG with 200 nm PJ34, MNNG followed by 5 mm NAD⁺, MNNG followed by 2.5 mm pyruvate, or medium exchanges only (control). Nuclei are counterstained with PI (red). Merged images show that DPQ, NAD⁺, and pyruvate block AIF translocation to the nucleus. B, C, Neurons transfected with NADase showed AIF translocation to the nucleus in both wild-type and PARP-1^−/− neurons fixed 4 h after BioPORTER transfection. The AIF translocation was blocked by 2.5 mm pyruvate. Images are representative of four independent experiments. Scale bar, 40 μm. D, E, Western blots show both total and mitochondrial AIF content at designated time points (hours) after transfection with NADase, with or without pyruvate. The Western blots were quantified after normalizing to either β-actin for total AIF, or the mitochondrial protein VDAC for mitochondrial AIF. Mitochondrial AIF release occurred in both wild-type and PARP-1^−/− neurons and was blocked by 2.5 mm pyruvate. F, G, Western blots show both total and mitochondrial AIF content at designated time points (hours) after placement in glucose-free medium, with or without pyruvate. Total AIF was quantified after normalizing to β-actin, and mitochondrial AIF was quantified after normalizing to the mitochondrial marker, VDAC. Mitochondrial AIF release occurred in both wild-type and PARP-1^−/− neurons and was blocked by 2.5 mm pyruvate. n = 3; *p < 0.01, **p < 0.001. Error bars indicate SEM.