Fig. 4.

GRAF1 promotes BAT differentiation by limiting RhoA/ROCK signaling. (a,b) 24 h following siRNA transfection, WT-1 pre-brown adipocytes were induced to differentiate into brown adipocytes over the indicated days using specific induction and differentiation factors (DM). Brown adipocyte differentiation was assessed by measuring UCP1 levels via Western blot (a) and quantified through densitometry(b). (c,d) RhoA activity in siRNA transfected WT-1 brown adipocytes was assessed by a standard GST-rhotekin precipitation assay(c) and quantified(d), n = 4 independent experiments. S1P denotes sphingosine-1-phosphate. (e) ROCK inhibitor Y27632 restored UCP-1 expression in GRAF1-depleted SGBS human brown adipocytes(n = 6/group). The cells were transfected with specified siRNA for 24 h and subsequently treated with either Vehicle(V) or the ROCK inhibitor Y27632(Y) during the brown adipocyte differentiation. siG1 refers to siGRAF1. Data are represented as mean ± SEM, ns not significant; *P < 0.05; **P < 0.01; ***<0.001 by two-tailed student’s t-test (b,d) or two-way ANOVA with post-hoc Fisher’s LSD test (e).