Fig 1: Brown adipocyte clones heterogeneously express brown, beige, and white fat markers.(A) mRNA expressions of Ucp1 at day 8 of differentiation (n = 2–5). (B) Heat map of mRNA expression for different adipocyte markers in all 67 clones, and in vitro differentiated white, beige and brown adipocytes at day 8 of differentiation (n = 3–8). Median expression for each cell line was transformed to log scale and gene wise z-scores were computed independently for each cell line. Dendrogram colors denote identified adipocyte clone cluster. (C) Correlation of Ucp1 with Pparg (left panel), Prdm16 (middle panel), and Ppargc1a (right panel). The values were mean ± SEM, and log transformed for Ucp1, Prdm16, and Ppargc1a (n = 4–5). Gene expression was normalized to Tbp. (D) Ucp1 expression of controls and cells treated for 3 h with 0.5 µM CL-316,243 (n = 2–5).Source data are available for this figure.
Fig 2: mRNA expression of brown, beige and white fat-specific markers in Eif5, Tcf25 and Bin1 knockdown clones and brown preadipocytes.(A, B) Semi-quantitative PCR analysis of Pparg, Ucp1, Prdm16, Ppargc1a, P2rx5, Cd137, and Asc1 expression at day 8 of differentiation and 3 h treatment with 0.5 µM CL-316,243 in (A) shScr B1 and shEif5 B1 (left panel), shScr D1 and shTcf25 D1 (middle panel), shScr D5 and shBin1 D5 (right panel) cells (n = 5), and (B) shScr, shEif5, shTcf25, and shBin1 on mixed brown adipocytes cells (n = 6). Data are shown as mean expression normalized to Tbp ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.Source data are available for this figure.
Fig 3: mRNA expression of brown and beige fat-specific markers in brown, beige and white adipocytes.(A, B) qPCR analysis of (A) Pparg, Prdm16, Ppargc1a, Ucp1, P2rx5, Pat2, Cd137, and Tmem26 expression in brown, beige and white adipocytes (n = 3–4) and (B) Adiponectin, Cd36, Fabp4 (log), Fasn, Glut4, and Hsl expression in brown adipose tissue clones (n = 2–3). All qPCR data were normalized to Tbp and shown as mean ± SEM. (B) *P < 0.05, **, ##P < 0.01, ***P < 0.001, ****P < 0.0001.Source data are available for this figure.
Fig 4: scRNAseq identifies distinct stages of brown adipocyte differentiation.(A) Immunofluorescence staining of UCP1 (green), F-actin (white), lipid droplets (red), and DAPI (blue) from WT and UCP1 knockout mice. (B) Quantifications of UCP1 content in individual brown adipocytes in brown adipose tissue normalized to total area (right panel, n = 11). Lipid droplets are included into area measured, and F-actin was used to distinguish each cell. (C) UMAP computed on full processed single-cell RNA-seq data set with annotated louvain clusters superimposed. (D) UMAP computed on set of preadipocytes with louvain subclustering. (E) Network of KEGG-enriched pathways of preadipocyte clusters. Significantly enriched pathways are connected to the respective cluster nodes. Size of the pathway nodes and proportion of the node-pie-chart refer to −log10 of enrichment P-value. (D) Node colors refer to single-cell cluster identified in (D). (F) Diffusion map dimensionality reduction of preadipocytes colored by Louvain clusters (top panel) and pseudotime (lower panel). (D, G) Expression of Pdgfra, Cd34, Cebpd, Cebpb, Cebpa, Pparg, Fabp4, and Lpl in preadipocytes shown in (D). The expression values are size factor normalized and log-transformed.Source data are available for this figure.
Fig 5: Expression of Ucp1 in adipose tissues and preadipocyte markers in the scRNAseq clusters of the SVF of BAT.(A) Quantitative PCR analysis of Ucp1 expression in interscapular brown adipose tissue, subscapular BAT, interscapular white adipose tissue, subcutaneous fat (SC), and perigonadal fat (PG) from 6-wk-old C57BL/6 mice (n = 3; ***P < 0.001). Data are mean expressions normalized to Tbp ± SEM. (B) Representative immunofluorescence staining of UCP1 on BAT used for quantifications in Fig 1B (Dapi: blue, UCP1: green, lipid droplets: red, F-actin: gray). (C) Violin plots showing the distribution of selected marker genes (Pdgfra, Pparg, and Fabp4) across louvain cluster computed on all cell types identified in the single-cell RNA-seq data set.Source data are available for this figure.
Supplier Page from Abcam for Anti-UCP1 antibody [EPR20381] (Alexa Fluor® 488)