Figure 5
From: Echinomycin inhibits adipogenesis in 3T3-L1 cells in a HIF-independent manner
Echinomycin suppresses C/EBPβ expression. (A) Immunoblot analysis of C/EBPβ protein in 3T3-L1 cells was performed at 0, 2, 6, 12, and 24 hr after MDI + DMSO or MDI + echinomycin (6 nM) treatment. C/EBPβ protein induction under MDI treatment was inhibited by echinomycin. Full-length blots are presented in Supplementary Figure. (B) The activity of the mouse Cebpb promoter was measured 8 hr after the treatments. Luciferase activity was normalized to CMV-renilla. *P < 0.05, **P < 0.01 vs. control (only-DMSO treatment), as determined by a two-way ANOVA with Bonferroni’s post-test. (C) (Left panel) HREluc reporter activity (firefly/TK-renilla) was measured at 16 hr after hypoxia treatment in stably expressing pX-330 against Hif1a. **P < 0.01 vs. pX330-control, as determined by a two-way ANOVA with Boneferroni’s post-test. (Right panel) Immunoblot analysis of C/EBPβ protein in 3T3-L1 cells that stably expresses pX330-control or pX330-Hif1a at 6 hr after MDI treatment. Knockout of Hif1a by pX330-Hif1a did not influence the induction of C/EBPβ by MDI treatment. Full-length blots are presented in Supplementary Figure. (D) (a) Mouse C/EBPβ stable overexpression 3T3-L1 clones were generated using a retroviral system and its C/EBPβ expression was confirmed by immunoblot analysis. Full-length blots are presented in Supplementary Figure. (b) Stable 3T3-L1 clones against control or C/EBPβ were treated with MDI + DMSO or MDI + echinomycin. Oil red O staining was performed at 8 day after induction. C/EBPβ overexpression restored adipogenesis under echinomycin treatment. For all panels, the data are the mean ± SEM or representative of at least three experiments.
