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. Author manuscript; available in PMC: 2017 Feb 9.
Published in final edited form as: Cell Metab. 2015 Dec 10;23(2):303–314. doi: 10.1016/j.cmet.2015.11.011

Figure 4. MiDAS Depends on AMPK Activation.

Figure 4

(A) Cells infected with control or shSIRT3-expressing lentiviruses (upper panel), or treated with DMSO, Rot, Anti A, or ethidium bromide (EtBr) (lower panel) for 10 days were analyzed for ADP:ATP ratios.

(B) WT or rho0 cells cultured + or −pyruvate and/or 2-DG for 10 days were analyzed for EdU labeling (upper panel) or SA-Bgal (lower panel).

(C) Control or shSIRT3-expressing cells were cultured for 10 days after infection. WT (mtDNA+) and rho0 (mtDNA−) cells were cultured + or − 1 mM pyruvate for 10 days, lysed, and analyzed by immunoblotting for total and phosphorylated AMPK and p53 and intracellular LMNB1, HMGB1, p21WAF1, p16INK4a, SIRT3, COX2, and actin.

(D) Control (siSIRT3/AMPK−), SIRT3 (siSIRT3+), and AMPKα-1/2 (siAMPK+) siRNAs were transfected into cells, which were analyzed by immunoblotting 4 days later for the indicated proteins.

(E and F) Cells infected with control or dominant-negative AMPK (AMPK-DN)-expressing lentiviruses, then control or shSIRT3-expressing lentiviruses were analyzed 7 days later for (E) SA-Bgal and (F) EdU incorporation. Bar graphs indicate mean + SEM.