Fig 2: Cysteine depletion induces browning of adipose tissue.a, Representative images of subcutaneous (SFAT) and visceral (VFAT) fat sections stained for UCP1 from Cth−/− mice fed CTRL or CysF diet for 6 days (scale bar, 100 μm). b, Representative H&E-stained sections of SFAT of Cth−/− mice fed CTRL or CysF diet for 6 days or CysF diet followed by Cys-supplemented diet for 4 days (CysF + Cys) (scale bar, 100 μm). c, Western blot detection of ATGL and UCP1 in SFAT from Cth−/− mice after 6 days of CTRL or CysF diet or Cys supplementation after CysF-induced weight loss. Actin is used as a loading control. d, qPCR analysis of thermogenic genes in SFAT of Cth+/+ and Cth−/− mice fed CysF diet for 6 days (n = 8 Cth+/+ and n = 10 Cth−/−). e,f, Faecal calorie content (n = 6 per group) (e) and cumulative food intake of Cth−/− mice fed CTRL or CysF diet for 4 days (f) (n = 6 per group). g, Linear regression analysis of EE against body mass during dark cycle at 4 and 5 days of weight loss (n = 10 Cth+/+ CysF and n = 12 Cth−/− CysF). h, Per cent body weight change of Cth−/− mice fed with CTRL diet or CysF diet (red line) for 5 days and then switched to Cys-containing diet (orange line) for 3 days (n = 6 per group). i, RER measured in metabolic cages, of Cth−/− mice fed with CTRL diet (n = 6) or Cys-containing diet after CysF-induced weight loss (n = 4). j, Average food intake of Cth−/− mice fed with CysF diet and then switched to Cys-containing diet for 2 days (n = 7 per group). Significance was measured with paired t-test. k, Linear regression analysis of EE against body mass during dark cycle of Cth−/− mice fed with CTRL (n = 6) or Cys-supplemented diet after CysF-induced weight loss (n = 4), average values of the first two nights after diet switch. l, t-SNE plot of scRNA-seq showing cluster identities from SFAT SVF from Cth−/− mice fed CTRL or CysF diet at day 4 of weight loss and bar chart showing population fold change (FC) in relative abundance of each cluster comparing Cth−/− CysF versus Cth−/− CTRL. DC, dendritic cell; APC, antigen-presenting cell. m, t-SNE plot displaying Pdgfra expression in red across all populations and Monocle analysis of clusters 0, 1 and 2, with colouring by pseudotime to show right most cluster giving rise to two separate clusters. Each cluster represented by colour in Cth−/− CTRL and Cth−/− CysF. Data are expressed as mean ± s.e.m. Statistical differences were calculated by two-way ANOVA with Sidak’s correction for multiple comparisons or unpaired two-tailed t-tests. NS, not significant.Source data

Fig 3: Phenotypic and transcriptomic analysis(A and B) Representative flow cytometry analysis of CAFs markers (CD26, FAPa, PDPN, PDGFRa, PDGFRb) on tumor cells and CAFs fractions (A). Normalized expression of each marker (B).(C) Analysis of CAFs, CD45+ TILs and tumor associated genes expression by RT-qPCR in each isolated fraction (n = 3 samples/fraction).

Fig 4: Gene expression analysis coupled with characterization of fibroblast and myofibroblast populations show that ZIPH-3c is associated with a unique, mildly fibrotic gene expression pattern.a, Principal component analysis (PCA) of a 50-gene panel from 4-week post-implantation samples plotted against each other. b, Genes associated with the negative direction of the third principal component (PC3). The expression of Tbx21 (T-bet) makes ZIPH-3c unique among the samples in the experiment. T helper cell-related genes are highlighted. c, Differentially expressed genes associated with the positive direction of the first principal component (PC1). All the genes have some connection to fibrotic diseases. Genes that are statistically significantly different from sham control and have a strong connection with fibrosis are highlighted. d, Total counts of fibroblasts associated with each implant, determined by flow cytometry 4 weeks post-implantation. Fibroblasts are defined as CD45− Lin− CD140a+, in which Lin consists of CD31, Ep-CAM, Tie-2, and Ter-119. e, Representative immunofluorescence microscopy images of tissues stained with collagen I, αSMA (myofibroblast), and F4/80 (macrophage). Locations of implants are marked by asterisks. Error bars, mean ± s.d. N=5–6 mice per treatment. One-way ANOVA was used for statistical comparison of multiple means. *, P < 0.05; **, P < 0.01; ***, P < 0.001; #, P < 0.00001.

Fig 5: AHR ligands upregulate genes associated with AHR pathway in cultured ASPCs.A: Schematic representation of experimental plan. Briefly, gWAT of WT mice was digested and SVF collected. ASPCs were isolated using FACS (CD45-CD31-PDGFRA+) and expanded in culture until confluent. ASPCs were then treated with FICZ (100 nM) and the conditioned media and lysates collected 3, 6 or 24 hr after treatment for downstream analysis. B: Cyp1a1 mRNA expression in cultured mouse ASPCs following treatment with vehicle or 100 nM FICZ as described in A, measured by RT-qPCR. Kruskal-Wallis test with Dunn’s multiple comparisons (n = 4). Each symbol represents an individual mouse; Error bars show SD. C: Heatmap of the RNA expression of the significantly upregulated DE genes in cells treated with DMSO or FICZ for 6 hr. Expression is normalized to the average expression of each gene. D: Protein-protein interaction map of the DE genes identified by bulk RNAseq, created using STRING (version 12.0). E: Ahr, Il6, Pdgfra, Pdgfrb and Pparg2 mRNA expression in sorted eYFP- and eYFP+ ASPCs from the gWAT of Cyp1a1 fate reporter mice injected with FICZ (10 mg/kg) five days before tissue collection, measured by RT-qPCR. Wilcoxon matched-pairs signed rank test (n = 6). The eYFP- and eYFP+ sample of each mouse is connected by a dashed line.