Figure 1.

Disrupting Rev-Erb expression accelerates myoblast differentiation. A) Expression of myogenesis genes in C2C12 myoblasts stably expressing Rev-Erbα/β shRNAs or control (GFP). shRNA expression was induced in 60% confluent cells at 24 h before RNA isolation (proliferating) or at 24 h or 96 h after stimulation of differentiation. B) Differentiation assay showing myotube formation (red) in C2C12 cells expressing Rev-Erb shRNAs after 4 days in differentiating medium. C) Quantification (n = 3) of nuclei/myotube or % differentiated cells as seen in images in (B). *p < 0.05 and ****p < 0.0001 as determined by two-tailed Students t-test. Data represented as mean and ±s.e.m. D) Structure of Rev-Erb agonist SR9011 and SR8278. E) Relative activity of SR8278 and SR9011 in Bmal-promoter driven luciferase reporter assay. F) Myogenesis gene expression in C2C12 cells treated with the Rev-Erb agonist SR9011 and G) antagonist SR8278. H) Immunoblot showing MYOD and Myosin heavy chain-2 (MHC2) expression in C2C12 treated with SR8278. I) Differentiation assay showing relative myotube formation (red) in SR8278 and SR9011 treated C2C12% differentiated cells and nuclear/myotube were quantified using imageJ (see Supplemental Figure S1). Representative images are shown. *p < 0.05 and **p < 0.01 as determined by two-tailed Students t-test. Data represented as mean and ±s.e.m.