Prolactin and Brown Adipocytes Figure 5. IGF-2 induces brown adipocyte proliferation and differentiation. A) Undifferentiated (d2) and differentiated (d7) T37i cells were treated with 20 nM IGF-2 or 20 nM insulin for 8 h. [3H]-thymidine (1 mCi per well) was then added to cells. Incorporation of [3H]-thymidine into DNA represents the mean6SE (three independent experiments performed in quadruplicate) Statistical significance: * p,0.05 and ** p, 0.01. B) T37i cells were grown in medium supplemented with 10% dextran-coated charcoal serum in the absence (control) or in the presence of 20 nM IGF-2 for 6 days. A full differentiation switch was observed with IGF-2. (Magnification X 20). Oil Red O staining was performed at d6 in the absence (base) or in the presence of 20 nM IGF-2, 20 nM insulin or 100 nM IGF-2. Oil Red O stained-cells were solubilized in 10% SDS and the OD520 measured. Results are mean6SE of three independent experiments. Statistical significance: *p,0.05, **p,0.01 and ***p,0.001, compared to untreated cells. Induction of PPARc2 mRNA expression in T37i cells treated or not by 20 nM IGF-2 or 20 nM insulin for 7 days. Quantitative real time PCR analysis of PPARc2 mRNA expression was performed, results expressed as attomol of PPARc2/fmol of 18S, represent mean6SE of three independent experiments performed in duplicate expressed as fold induction vs undifferentiated cells (*p,0.05 and *** p,0.001). C) PRLR KO preadipocyte cells were grown in medium supplemented with 10% dextran-coated charcoal serum without or with 20 nM IGF-2 for 6 days where the appearance of fully mature adipocyte foci were observed (Magnification620). doi:10.1371/journal.pone.0001535.g005 PLoS ONE | www.plosone.org 8 February 2008 | Issue 2 | e1535