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. Author manuscript; available in PMC: 2015 May 3.
Published in final edited form as: Endocrinology. 2005 Oct 6;147(1):141–154. doi: 10.1210/en.2004-1649

Fig. 8.

Fig. 8

Effect of constitutive activation of AR on adipogenesis. A, Expression of C/EBPα mRNA and protein. Cells were transiently transfected either with pAct-AR or pARE4-Luc using Lipofectamine 2000 and allowed to differentiate in adipogenic conditions for 9 d in absence of androgen. C/EBP-α protein and mRNA were analyzed by Western blot (right) and real-time RT-PCR (left), respectively. Data for real-time RT-PCR (done in quadruplicate for each treatment) and Western blot are from a single experiment representative of three separate experiments. B, Dual-luciferase reporter assay. Cells were transiently transfected with either pGL3, pARE4-Luc alone or pAct-AR in combination with pARE4-Luc and allowed to grow for 2 d in growth medium. Cells were also cotransfected with Renilla luciferase plasmid pRL-TK-Luc (50:1) using Lipofectamine 2000 and dual-luciferase assay was performed using standard protocols. The relative luciferase activity is presented in arbitrary units as mean ± sd from four separate experiments. C, Immunofluorescence analysis. Cells plated on two-well chamber slides were transiently transfected with pAct-AR, fixed with 2% paraformaldehyde, and immunofluorescence was performed using anti-β catenin antibody (Texas Red). D, Expression of p21 protein. Cells were transiently transfected either with pARE4-Luc or pAct-AR as described in panel A and allowed to differentiate in adipogenic medium for another 2 d. Cells were lysed, and p21 expression was analyzed using anti-p21 antibody.