Fig. 3

NAD⁺ synthesized via AMPK and NAMPT pathways stabilizes HIF-1α during hypoxia. (A and B) NAD⁺-sensitive HIF-1α accumulation. HIF-1α protein levels were determined under NAD⁺-reduced and -reconstituted conditions after hypoxic exposure for 9 h; NAD⁺ limitation was achieved via use of NAMPT- or AMPK-siRNA whereas NAD⁺ reconstitution involved external addition of 1 mM NAD⁺ (A) or nicotinic acid (NA) (B). (C) NAD⁺/NADH-sensitive recovery in HIF-1α levels. Pyruvate or NAD-mediated recovery in HIF-1α levels was measured under NAD⁺-reduced and NAD⁺-reduced-plus-NADH-upregulated conditions, upon hypoxic exposure for 9 and 24 h, respectively; tests employed AMPK-siRNA treatment. (D) The need for SIRT1 expression in terms of NAD⁺/NADH-sensitive HIF-1α recovery was evaluated in HeLa cells in which SIRT1 was or was not stably depleted, using SIRT1- or control-shRNA, respectively. Statistical significance (p-value) was determined using the ANOVA-t-test. * : p < 0.05, ** : p < 0.01