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. Author manuscript; available in PMC: 2009 Feb 2.
Published in final edited form as: J Cell Biochem. 2008 Sep 1;105(1):89–98. doi: 10.1002/jcb.21801

Fig. 6.

Fig. 6

Effects of shRNA-mediated HIF-1α knockdown on the ability of PGF2α to inhibit MDI-induced 3T3-L1 preadipocyte differentiation. A: Demonstration of decreased HIF-1α protein expression after HIF-1α shRNA-mediated knockdown. 3T3-L1 preadipocytes transduced with either control pSUPER retrovirus or the pSUPER-shHIF-1α retrovirus were treated with MDI in presence of 100 nM PGF2α. At day 2 post-differentiation nuclear extracts were prepared and analyzed by immunoblotting for expression of either HIF-1α or Sp1 as a control. B: RT-PCR analysis of DEC1 expression in either control or HIF-1α shRNA-transduced 3T3-L1 preadipocytes treated with MDI in the presence of 100 nM PGF2a for 0, 1, or 2 days. The expression of HPRT is shown as a control. C: Representative cell morphologies of either control or HIF-1α shRNA-transduced 3T3-L1 preadipocytes induced to undergo differentiation by the standard MDI protocol in the additional presence of 100 nM PGF2α for the first 48 h of the culture period. At 10 days post-differentiation cell morphology was analyzed by light microscopy. D: Effects of HIF-1α shRNA-mediated knockdown on the dose-dependent ability of PGF2α to inhibit adipocyte differentiation. 3T3-L1 preadipocytes transduced with either control pSUPER retrovirus or the pSUPER-shHIF-1α retrovirus were induced to undergo adipocyte differentiation by the standard MDI protocol in the presence of a range of PGF2α concentrations (6.25, 12.5, and 25 nM) present during the first 48 h of the culture period. After 10 days the extent of adipocyte differentiation was determined by Oil Red O-staining. E: Effects of HIF-1α shRNA-mediated knockdown on the ability of PGF2α to inhibit the expression of adipocyte specific marker proteins. 3T3-L1 preadipocytes transduced with either control pSUPER retrovirus or the pSUPER-shHIF-1α retrovirus were induced to undergo adipocyte differentiation by the standard MDI protocol in the presence of 12.5 nM PGF2α for the first 48 h of the culture period. After 10 days, whole cell extracts were prepared and the expression of C/EBPα, PPARγ, aP2 and actin was determined by immunoblotting. Data are representative of three independent experiments.