Experimental strategy used for large-scale gene expression profiling of mESC adipogenesis. (a) Summary scheme of our experimental design. Adipocyte commitment was selectively stimulated through exposure of EBs to CD2314, or repressed through the addition of the GSK3 inhibitor BIO, or both compounds, between day 3 and day 6. Adipocyte terminal differentiation was further induced by addition of the adipogenic compounds insulin (Ins), triiodothyronine (T3), and rosiglitazone (BRL) from day 7 to day 21. For microarray analysis, samples were generated before (day 3), right after (day 6), or 5 days after (day 11) exposure to control medium, CD2314, BIO, or CD2314+BIO and analyzed using Mouse Genome 430 2.0 Affymetrix Arrays. Expression of adipocyte differentiation markers could first be detected at day 11, while mature adipocytes were detected at day 21. (b, c) Quantification of Fabp4 or Lpl RNA expression by quantitative PCR at day 11. The relative expression level of each RNA upon CD2314 stimulation was considered as 100%. (d) Oil red O staining of mature adipocyte colonies at day 21. Scale bar: 50 μM. (e) Quantification of the percentage of EB outgrowths with adipocyte colonies at day 21. (f) Quantification of glycerol-phosphate dehydrogenase (GPDH) activity at day 21. Here and in the following figures, data are displayed as mean values ± standard error of the mean of at least three independent experiments.