Figure 3: Interaction between G-actin and MKL1 and the consequent cytoplasmic sequestration of MKL1 are required for adipocytic differentiation in DFAT cells.

(a) Cells stably expressing 3 × FLAG-tagged ER (ER-MOCK, control), ER-MKL1 or ER-MKL1-N100 were exposed for 48 h to inducers of adipogenic differentiation in the absence or presence of TAM (1 μM), after which nuclear (N) and cytoplasmic (C) fractions were prepared from the cells and subjected to immunoblot analysis of FLAG, lamin C (nuclear marker) and MEK1/2 (cytoplasmic marker). (b) Cells treated as in a were analysed for the relative abundance of Pparg mRNA or subjected to immunofluorescence analysis of FLAG and PPARγ. Nuclei were stained with Hoechst 33342. Scale bars, 50 μm. (c) Relative abundance of Cebpa, Fabp4, Slc2a4 and Plin1 mRNAs in cells treated as in a. (d) Cells treated as in a were cultured for an additional 48 h (total of 96 h) and then stained with oil red O. Scale bars, 100 μm. The A₅₁₀ of dye extracted from the stained cells was also determined. n.s., not significant. (e) Cells stably expressing ER-MKL1 or ER-MKL1-N100 were exposed for 24 h to Lat A (0.4 μM) in the presence of TAM (1 μM) and then subjected to immunofluorescence analysis of FLAG and PPARγ (upper panels). Nuclei were stained with Hoechst 33342. Scale bars, 50 μm. At least 300 cells per coverslip were scored for determination of the percentage of the subcellular localization of MKL1 (centre panel). N, nuclear; N/C, comparable intensity in nucleus and cytoplasm; C, cytoplasmic. (f) Relative abundance of Pparg, Cebpa, Fabp4, Slc2a4 and Plin1 mRNAs in cells treated as in e. All quantitative data are means±s.d. (n=3 experiments). *P<0.05, Student’s t-test.