Figure 1: HO-1 is a target of HFD in adipose precursors.

All visceral and subcutaneous APs were isolated from mice on a SD or HFD for 3 days via Sca-1 bead pull down (see ‘Experimental Procedures’). (A) Dot plot displaying diet-induced changes in gene expression in APs. Purple, green and red spots represent genes significantly differentially expressed greater than 2-fold (P < 0.05; n = 6 mice, pooled into two replicates per group). Grey dots indicate genes that are not statistically significantly enriched. (B) Venn diagram illustrating overlap between gene signatures derived from expression analysis of AP fractions. (C) GO analysis using DAVID⁸⁰ of scAP and viAP molecular signatures. Notable terms are highlighted; (see also Supplemental Tables S3 + S4). P-values are derived from Fisher’s exact test and pass Benjamini-Hochberg corrected P < 0.05. (D) GSEA analysis of AP fractions. Analyzed gene sets include GO, KEGG and Reactome-derived cell cycle pathways as well as custom-curated genesets from a 3T3-L1 differentiation time course³¹ (see ‘Experimental Procedures’ for details). (E) Upper panel: Network plots for genes comprising the ‚Leading edge‘ of cell cycle from the KEGG, GO and Reactome compendium. Lower panel: Network plots for the 66 genes comprising a core HFD-associated AP gene signature. Blue node color represents downregulation and red node color represents upregulation of gene expression in APs. The node size is associated with the gene’s co-expression in the entire data set. The edge (line) thickness is linked to the gene’s connectivity (co-expression within the module). (F) Heatmap of top 10 genes regulated by HFD common to APs from scWAT and viWAT. Each column represents pooled cells from three mice. Blue color represents downregulation and red color represents upregulation of gene expression. (G) HO-1 mRNA expression in scWAT or viWAT fractions (n = 6). (H) Western blot of protein lysates from APs enriched from SVF of scWAT or viWAT. Each lane represents pooled cells from 2 mice. (I) Quantification of western blots in (H) showing HO-1 protein levels normalized to β-actin. (J) Whole mount immunofluorescence staining for HO-1 and CD24 in viWAT. Arrowheads indicate CD24+ cells coexpressing HO-1. Bar = 20 μm.