Figure 4: Loss of MKL1 is sufficient to drive adipocyte differentiation in vitro and in vivo.

(a) DFAT cells were transiently transfected with MKL1 (siMkl1-a or -b) or control (siControl) siRNAs for 48 h and then cultured in growth medium without an adipogenic cocktail for 96 h, after which the relative abundance of Pparg, Fabp4, Slc2a4 and Plin1 mRNAs was determined (upper panels). The cells were also subjected to immunofluorescence analysis of PPARγ expression (bottom left panel). Nuclei were stained with Hoechst 33342. Scale bars, 100 μm. They were also stained with oil red O (bottom centre panel; Scale bars, 100 μm), and the A₅₁₀ of dye extracted from the stained cells was determined (bottom right panel). (b) NIH 3T3 fibroblasts were transiently transfected with MKL1 (siMkl1-a) or control siRNAs for 48 h and then cultured in growth medium without an adipogenic cocktail for 96 h, after which the relative abundance of Pparg, Fabp4, Slc2a4, and Plin1 mRNAs was determined (left panels). The cells were also subjected to immunofluorescence analysis of PPARγ expression (right panel). Nuclei were stained with Hoechst 33342. Scale bars, 100 μm. (c) DFAT cells were transiently transfected with MKL1 (siMkl1-a) or control siRNAs for 48 h and then injected subcutaneously into mice. Serial sections of the injection site were subjected to immunohistochemical staining of GFP (to identify injected cells) as well as of FABP4 and perilipin (markers of terminal adipogenic differentiation) at 2 weeks after injection. The sections were counterstained with hematoxylin. Data are representative of five mice per group. Scale bars, 100 μm. All quantitative data are means±s.d. (n=3 experiments). *P<0.05, Student’s t-test.