Figure 1: Disruption of actin stress fibres is required for adipocyte differentiation.

(a) Fluorescence microscopy of the actin cytoskeleton (stained with phalloidin) as well as of PPARγ or perilipin expression during adipogenesis in DFAT cells. Nuclei were stained with Hoechst 33342 (blue fluorescence). Scale bars, 20 μm. (b) Relative abundance of Pparg mRNA during adipocytic differentiation in DFAT cells (left panel). (a–f) P<0.05, Tukey’s honest significant difference test. A nuclear fraction prepared from the cells was also subjected to immunoblot analysis of PPARγ and lamin C (loading control) (right panel). (c) Time-lapse imaging of DFAT cells expressing red fluorescent protein (RFP)–tagged actin at the indicated times (hours:minutes). Scale bars, 50 μm. (d) 3T3-L1 preadipocytes transfected with cofilin1 (siCfl1-a) or control (siControl) siRNAs were exposed to inducers of differentiation for 96 h. They were then subjected to fluorescence microscopic analysis of the actin cytoskeleton and PPARγ expression. Nuclei were stained with Hoechst 33342. Scale bars, 50 μm. Relative abundance of Pparg, Cebpa, Fabp4, Slc2a4 and Plin1 mRNAs. *P<0.05, Student’s t-test. (e) Oil red O (ORO) staining of cells treated as in d. Bars, 100 μm. The absorbance at 510 nm (A₅₁₀) of dye extracted from the stained cells was also determined. *P<0.05, Student’s t-test. (f) Immunoblot analysis of active and total forms of Rho proteins during adipocyte differentiation in DFAT cells (left panels). GAPDH was examined as a loading control. Quantification of the immunoblotting data was performed using densitometry. Data were normalized to the amount of total Rho (right panel). *P<0.05, Student’s t-test. n.s., not significant. (g) Fluorescence microscopy of the actin cytoskeleton as well as of PPARγ or perilipin expression in DFAT cells expressing HA-tagged RhoAV14 or RhoAN19 and exposed for 96 h to inducers of differentiation in the absence or presence of Y-27632 (30 μM) or CytD (0.2 μM). Nuclei were stained with Hoechst 33342. Scale bars, 20 μm. At least 50 HA-positive cells were scored for determination of the percentage of those expressing PPARγ. *P<0.05, Student’s t-test. All quantitative data are means±s.d. (n=3 experiments fibres).