Figure 6. HIF1α-dependent autocrine/paracrine PDGFR signaling drives PPARγ S112 phosphorylation and inhibition of adipogenesis in PDGFRβ+ mural cells.
(A) Experimental design: iWAT PDGFRβ+ cells from Control or Mural-Hif1aDN mice were treated with vehicle or 50 μM DMOG (48 hours). Washed cells were transferred to transwell membranes (Top layer cells) overlaying stable Mural-PPARγ2 cells (without DMOG) (Bottom later cells). Co-cultures were maintained in serum free media (+/− PDGFR antagonist) for 6 hours before bottom layer cells were harvested for western blot analysis. Parallel co-cultures were established to assay for adipogenesis of Mural-PPARγ2 cells, with PDGFR antagonist or vehicle added to the adipogenic induction media for the first 48 hours (See Material and Methods for additional details).
(B) Western blot of indicated proteins in Mural-PPARγ2 cells following culture with the indicated cells/treatments.
(C) Oil Red-O staining 7 days after inducing adipogenesis of Mural-PPARγ2 cells exposed to the indicated cells/treatments. Scale bar denotes 200 μm.
(D) Western blot of indicated proteins in Mural-PPARγ2 cells following culture with the indicated cells/treatments.
(E) Oil Red-O staining 7 days after inducing adipogenesis of Mural-PPARγ2 cells exposed to the indicated cells/treatments. Scale bar denotes 200 μm.