Fig 2: Bulk RNA-seq of flow-sorted APC CD24+ progenitors and CD24-preadipocytes reveals transcriptomic signatures for adipogenic, mitochondrial, and energetics pathways by low n6/n3 FA ratios(A) Differential gene expression by n6/n3 exposure group within either progenitor or preadipocyte APC subtypes was analyzed using comparative analysis in Ingenuity Pathway Analysis. Significantly enriched canonical pathways in common between CD24+ progenitor and CD24-preadipocyte APCs were ordered by FDR p value (=0.01) and colored by the predicted activation Z score. Coloring indicates common pathways predicted to be “on” (yellow), “off” (navy), or “no prediction” (white) due to low n6/n3 exposure. Pathway activation Z score values are presented for either APC subtype, and values =2.0 or = -2.0 are considered significant.(B) Significantly different genes in either CD24+ progenitor or CD24-preadipocyte relative to the high n6/n3 FA ratio group colored by Log2 fold change and grouped into transcription factor genes (Regulatory) and signal transduction pathway genes (Ligands/Kinases).(C) Significantly different lipid metabolism, mitochondrial, and glycolytic genes indicating that the low n6/n3 FA ratio changes oxidative capacity potentially through aldehyde dehydrogenases (Aldh1a1 and Aldh1a3), uncoupling protein 2 (Ucp2), citrate synthase (CS), isocitrate dehydrogenase (Idh1 and 2), malic enzyme (Me1), malate dehydrogenase (Mdh1), phosphofructokinases (Pfkm, Pfkp, and Pfkfb1), and transketolase (Tk) (See also Figure S2).(D) Canonical Pathway enrichment within either CD24+ progenitor or CD24-preadipocyte APCs plotted by the percentage DE-Gs within a pathway (%) by the pathway enrichment significance (-log(p value)) and colored by the predicted pathway activation (Z score, >2.0 and < -2.0 was considered significant) (See also Data S3 for all pathways).(E) Immunoblots for NR2F2, UCP2, C/EBPa, and DNMT1 from all lineage negative, CD29+/CD34+, and Sca1+ APCs isolated by flow cytometry from pooled litters (n = 3 litter dyads per n6/n3 FA ratio group; litters standardized to 6-8 pups/litter).(F) EVC005 murine APC cell line was treated with and without NR2F2 ligand 1-DSO for 48 h, which induced gene expression of Prdm16, P2rx5, Runx1, Ucp2, Dnmt1, Ppar?2, and C/ebpa.(G) TMRE live cell mitochondrial potential dye following 48 h of 1-DSO treatment in EVC005 cells, indicating an increased mitochondrial potential. Panels F and G are n = 3 wells per treatment group. Scale bar represents 50 µm. Data are represented as mean ± SEM.

Fig 3: Overexpression of SOX4 in preadipocytes diminished the pool of preadipocytes under HFD condition3-week-old WT and Pref1-Sox4 mice were housed at room temperature and fed with NCD or HFD for 20 weeks.(A) Pref1-Sox4 mice were generated by directly inserting coding sequence (CDS) sequence of Sox4 after a 6-kbp fragment upstream of the transcriptional start site of the Pref1 promoter.(B) iWAT SVFs were isolated from NCD or HFD-fed WT and Pref1-Sox4 mice and cultured for 2 h to exclude the non-adherent cells. Then, the cultured cells were collected and the expression of Sox4 was detected by qPCR (n = 4–6). mRNA levels were normalized to 18s RNA. The mean CT was converted to relative expression value by the equation 2-?Ct.(C) The expression of SOX4 in iWAT SVFs of HFD-fed WT and Pref1-Sox4 mice was detected by Western blotting.(D) The expression of Sox4 in indicated cell populations sorted by flow cytometry was examined by qPCR (n = 4). mRNA levels were normalized to 18s RNA. The mean CT was converted to relative expression value by the equation 2-?Ct.(E–G) The proportions of CD45-: CD31-: Ter119-: PDGFRa+ (Lin-: PDGFRa+ cells) adipocyte precursors (E), DPP4+ MSCs (F) and ICAM1+ preadipocytes (G) were analyzed by flow cytometry in NCD- and HFD-fed WT and Pref1-Sox4 mice (NCD group: n = 4; HFD: n = 4–5).(H) The proportions of MSCs with adipogenic potential (Lin-: CD34+: CD29+), adipocyte progenitors (Lin-: CD34+: CD29+: Sca-1+: CD24+, CD24+ cells), committed preadipocytes (Lin-: CD34+: CD29+: Sca-1+: CD24-, CD24- cells), and nonadipogenic fibroblasts (Lin-: CD34+: CD29+: Sca-1-, Sca-1- cells) were analyzed by flow cytometry in NCD- and HFD-fed WT and Pref1-Sox4 mice (NCD group: n = 3; HFD: n = 4–6).3-week-old WT and Pref1-Sox4 mice with HFD for 20 weeks were used in this study.Data are represented as mean ± SD. ns, no significance, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.