Loss of Sdc1 ablates adipocyte differentiation <i>in vitro</i>.

<p><b>A</b>. 3T3-L1 cells (pre-adipocytes), 3T3-L1 cells 8 days after initiation of the differentiation protocol (adipocytes) and 3T3-L1 cells after knockdown of Sdc1 with siRNA were assayed for Sdc1 expression by immunofluorescent antibody staining. <b>B</b>. A similar series of cultured cells were stained with Oil Red-O, a dye that dissolves in lipid drops accumulating in differentiated adipocytes. <b>C</b>. Cell lysates were assayed for markers of differentiation by qPCR (PPARγ, peroxisome proliferator-activated receptor gamma; FABP4, fatty acid binding protein-4; LPL, lipoprotein lipase; FASN, fatty acid synthase; CD36, thrombospondin receptor) and by Western blotting (FasN, Cd36, activated phospho-IRS1). <b>D</b>. Ear mesenchymal stem cells (eMSCs) were isolated from <i>Sdc1−/−</i> and BALB/c mice, transferred to culture, and induced to differentiate. Differentiation was visualized by Oil Red-O staining. Corresponding nuclear stains (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004514#pgen.1004514.s006" target="_blank">Fig. S6C</a>) illustrate that the cell densities are approximately similar. <b>E</b>. To evaluate the impact of a heparan sulfate mimetic, heparin, on the ability of 3T3-L1 preadipocytes (A,B,C) and adipocytes (D,E,F) to take up VLDL, cells were incubated in presence (B,E) or absence (A,C,D,F) of 200 µM heparin with di-I labeled VLDL for 3 hours at 37°C. Fields from A and D were magnified (C and F respectively) to show the size and morphology of the red-stained vesicles. Uptake was quantified (right hand side panel). Lower concentrations of heparin showed similar effects (50 µM; data not shown).