Figure 2.

(A) Human preadipocytes were induced to differentiate for 96 h with rosiglitazone, insulin, dexamethasone, and IBMX (MIX) in the presence or absence of 10 nM DHT. Cells were then stained with DAPI (DNA), CellMask Blue (CMBl, general protein dye), and LipidTOX (Lipid), followed by HTM. Scale bar is 50 μm. (B) Lysates were prepared from adipocytes differentiated for 4 d in the presence or absence of 10 nM DHT. Protein levels of AR and PPARγ were detected by western blot. (C) Human AR-FLAG (fAR) or vector control lentiviral particles were prepared and introduced into 3T3-L1 preadipocytes for 48 h before chemical induction of differentiation. Human AR expression was detected in both human adipocytes and 3T3L1 expressing fAR before and after 96 h of differentiation. (D) After cell differentiation in the presence or absence of 10 nM DHT for 96 h, cells were processed for immunofluorescence to AR, stained for lipid and DAPI/CMBl, and imaged. Scale bar, 20 μm. (E) PPARγ2 and (F) FABP4 mRNA levels were measured from 3T3L1 cells expressing vector (pcDH) or fAR differentiated in the presence or absence of 10 nM DHT for 96 h. (n=2 independent experiments +/− s.e.m., #p<0.05 compared to no differentiation or *p<0.05 compared to differentiation without DHT). (G) Lipid accumulation was quantified by HCA after lentiviral expression of human AR (n=average of 4 wells+/− s.e.m.) or androgen treatment in human preadipocytes (n=5 independent experiments+/− s.e.m., *p<0.05).