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. 2010 Jun 14;30(16):4035–4044. doi: 10.1128/MCB.00175-10

FIG. 5.

FIG. 5.

NO-induced downregulation of PGC-1α is mediated by Foxo3a. (A) BAEC were infected with TM-Foxo3a (MOI, 10) or control adenovirus and, 24 h later, were incubated with DETA or DETA-NO (62 μM, 12 h) as indicated. (Top) PGC-1α mRNA expression. Control samples (DETA) were assigned the value of 100%. (Bottom) PGC-1α WB. (B) BAEC were incubated with DETA or DETA-NO (62 μM, 12 h), and Foxo3a binding to three positions within the PGC-1α promoter was analyzed by ChIP. IP DNA was analyzed by qPCR. The 18S RNA gene was used as a negative control. (C) BAEC were transfected with 500 ng of the PGC-1α promoter vector and, 24 h later, incubated as indicated with DETA-NO (62 μM), 8-Br-cGMP (100 μM), and LY294002 (10 μM) for 12 h. (D) BAEC were cotransfected with the PGC-1α promoter vector (500 ng) and TM-Foxo3a (1 ng) and treated as indicated with DETA-NO. (E) BAEC were transfected with the PGC-1α promoter or with the IRS mutant vector (500 ng). Control samples (DETA) were assigned the value of 100%. (F) Scratch assay using serum-starved confluent BAEC. Cells were infected with Foxo3a shRNA (shFoxo3a) or control adenovirus (MOI, 25) at 24 h before the beginning of the assay and then treated with 100 μM 8-Br-cGMP as indicated. Cells were allowed to migrate for 18 h. Control samples (control shRNA [shC] plus vehicle) were assigned the value of 100%. (G, H) MitoSOX Red labeling of fixed BAEC infected with shFoxo3a (G), TM-Foxo3a (H), or the corresponding control adenovirus and treated with 100 μM 8-Br-cGMP for 6 h. #, P < 0.05 versus paired control.