(A) PPARγ-tTA;TRE-Cre;R26R (PPARγ-R26R) or PPARγ-tTA;TRE-H2B-GFP (PPARγ-GFP) (bottom panels) mice were treated with or without Dox either from embryonic day 0 (E0) to postnatal day 30 (P30) (top row) or from P2 to P30 (middle and bottom rows) and then inguinal and retroperitoneal white adipose tissues (IWAT and RWAT respectively) were excised and examined for lacZ (blue) or GFP (green) expression. Left panels show equivalent depots of control mice containing either TRE-Cre;R26R or TRE-H2B-GFP.
(B) P30 stromal-vascular (SV) cells from wild-type (left panel), PPARγ-R26R (middle panel) and PPARγ-GFP (right panel) WAT were examined for expression of PPARγ (red) with immunocytochemistry (left panel) or for reporter expression. Left panel: Nuclei stained with DAPI (blue). Yellow arrows indicate cells that express PPARγ (purple). Middle panel: lacZ (blue), nuclei counterstained with nuclear fast red (red). Right panel: GFP (green), nuclei stained with DAPI (blue).
(C) Flow cytometry profiles of SV cells of untreated TRE-H2B-GFP (left) or PPARγ-GFP mice treated without (middle) or with Dox (right) from P2 to P30. X-axis is GFP fluorescent intensity. Y-axis is PE channel to help illustrate the distribution of GFP+ cells. Inset: X-axis is GFP fluorescent intensity; Y-axis is the cell count of the GFP+ cells per interval of fluorescent intensity (one unit = 1000). SV cells from TRE-H2B-GFP mice served as a gating control.
(D) SV cells removed from TRE-Cre;R26R (left panel) and PPARγ-R26R mice treated as indicated were isolated and stained with X-gal (blue) and nuclear fast red (red). Dox treatment did not alter the number or percentage of lacZ+ cells based upon statistical analysis of >2,000 cells counted in each group.
Scale bars: 2mm (A), 50μm (B, D)

(A) GFP- and GFP+ SV cells from PPARγ-GFP mice were sorted, plated, cultured to confluence and insulin-stimulated adipogenesis was examined with the lipid-specific stain Oil Red O (red).
(B) Sorted GFP+ SV cells were cultured in media or media supplemented with insulin and then stained with Nile Red, a lipid-specific fluorescent dye, to simultaneously visualize fat accumulation and GFP expression.
(C) Left panel: qPCR analysis of the indicated markers in sorted GFP+ cells before (green bars) and after (blue bars) insulin-stimulated adipogenesis. C/EBPα is an adipogenic transcription factor; leptin, adiponectin (ADPN), and adipsin are adipokines; Pref-1 is a preadipocyte marker whose expression inversely correlates with adipogenesis. Right panels: SV cells from P30 PPARγ-GFP adipose depots were examined for GFP (green) and perilipin (red, an adipocyte marker) expression both before (top) and after (bottom) adipogenic induction. Nuclei stained with DAPI (blue).
(D, E, F) FACS-isolated GFP+ SV cells were implanted into nude mice and the tissue that formed after 1 month was photographed with bright field (D, left panel) and fluorescent microscopy (D, right panel) and examined with H&E staining (E, left panel), GFP fluorescence and Nile Red staining (E, right panel), and GFP fluorescence and perilipin immunochemistry (F).
(G) Left panel: P30 PPARγ-GFP adipose depot SV cells were examined for Sca-1 and GFP expression with flow cytometry. The box indicates the Sca1⁺GFP⁺ double positive population. Right panels: IWAT (adipose, top) and SV cells (bottom) of aP2-GFP transgenics were analyzed for GFP expression, which was present in adipocytes but not SV cells. PPARγ-GFP serves as a control. Nuclei stained with DAPI (blue).
(H) qPCR analyses of the indicated markers of FACS-isolated GFP+ SV cells (green bars) and floated adipocytes (blue bars).
(I) GFP- SV cells, GFP+ SV cells and adipocytes were subjected to gene expression profiling. The heatmap illustrates 152 genes that differentiate GFP+ SV cells from the other populations. The red depicts a ≥2-fold increase in gene expression while the green depicts a ≤2-fold decrease in gene expression.
Scale bars: 50μm (A-C, E, G right panel bottom row), 2mm (D, G right panel top row), 20μm in confocal images (F).

(A) SVP structures from P30 PPARγ-GFP mice were photographed with light (left panel) and fluorescent (right panel) microscopy. Arrows indicate SV tube.
(B) PPARγ-GFP SVP tubes were isolated and stained with the lipid-specific dye BODIPY either prior to culture (left panel) or after 3 days cultured on petri dishes in insulin (right panel). Arrows indicate SV tube.
(C) PPARγ-GFP SVP tubes were cultured in suspension. Formation of adipocytes that derive from the GFP+ tubes was assessed with BODIPY staining (red). GFP (green). Lipid droplets visualized with confocal microscopy (right panel).
(D) SVP isolates of P30 PPARγ-GFP mice were examined for expression of GFP (green) and the indicated endothelial (PECAM, red) and mural cell (SMA and NG2, blue) markers.
(E) PPARγ-GFP SVP vessel examined for expression of GFP (green), and the mural cell markers PDGFRβ (red) and SMA (blue). Yellow arrows indicate position of GFP+ nuclei within mural cells.
Scale bars: 50μm (A, B, left panels of C), 20μm in confocal images (right panels of C; D, E).

(A) P30 PPARγ-GFP WAT was freshly frozen, cryo-sectioned, and examined with direct fluorescence for GFP and indirect immunofluorescence for the indicated endothelial (PECAM, red) and mural cell (SMA, blue; NG2, blue; PDGFRβ, red) markers.
(B) Cryo-section of a PPARγ-GFP adipose depot showing expression of GFP, PDGFRβ (red) and SMA (blue). Arrows indicate some mural cell nuclei that express GFP. (C) Muscle cryo-sections and retinal whole mount of PPARγ-GFP mice were examined for GFP, PECAM, and SMA as in (A, B). GFP was not expressed in mural cells of these tissues.
(D) RWAT (top panels, 5x) and IWAT (bottom panels, 20x) of P30 PDGFRβ-Cre;R26R and SM22-Cre;R26R mice were stained for β-galactosidase expression (blue).
(E) SV cells were isolated from P30 wild-type mice and sorted with a PDGFRβ antibody. Top panels: confluent PDGFRβ negative and positive cells were cultured in insulin and fat formation assessed with BODIPY (red). Bottom panels: PDGFRβ negative and positive cells were transplanted into nude mice, and the resultant tissues were sectioned and H&E stained.
(F) Adipose SVF and cells dissociated from kidney were sorted with a PDGFRβ antibody, and PDGFRβ positive cells were cultured in the absence (top) or presence of TZD (bottom).
Scale bars: 20μm in confocal images (A-C), 1mm (D), 50μm (E, F).