Fig 2: Afadin deletion leads to transcriptomic alterations in BAT at thermoneutrality and in response to cold. Male fat-specific Afadin knock-out (FAKO) and control littermates (CTRL) were housed at thermoneutrality (29 °C) for 2 weeks followed by 5 °C cold-exposure for 6 h and BAT was harvested and RNA-sequencing was performed. (a,b) Gene set enrichment analysis (GSEA) for each comparison. Top 10 significant up/down regulated KEGG terms containing at least 10 genes were plotted using gene ratio, regulated by temperature (a) and in between genotypes (b). Full list of enriched KEGG terms can be found in Supplementary Table 1.

Fig 3: Afadin deletion modulates the transcriptomic profile within specific pathways regulating BAT function. (a,b) KEGG term—gene interaction plot depicting the linkages of genes and biological concepts (KEGG terms) as a network. Log2 fold changes (FAKO vs CTRL) of gene expression within selected enrichments at 29 °C (a) or at 5 °C (b).

Fig 4: Tsc1-deficient spheres display altered Afadin/Cadherin-based adhesion system localization.a Representative images of 5-day mIMCD3s spheres of the indicated genotypes immunolabelled with Afadin (red), E-cadherin (green), γ-Tubulin (magenta) antibodies, and DAPI to show the relative localization of Afadin and E-cadherin in interphase and relative to the mitotic spindle poles in 3D spheroids. Insets show higher magnification of one mitotic cell per genotype, to show the relative localization of Afadin and E-cadherin in mitotic cells. In the insets, yellow arrows indicate the localization of the Afadin spots. White arrows indicate localization of the poles of the mitotic spindles. A dotted yellow line indicates Afadin staining in the whole apical domain of mitotic cells. Scale bar, 10 µm. b Percentage of mitosis showing one Afadin spot adjacent to each spindle pole (white), or perturbed afadin localization adjacent to a single spindle pole (dark gray). An average of 10 mitosis were analyzed per experiment per each genotype. The histograms are an average of three independent experiments. Mean ± SEM. c Representative images of 5-day mIMCD3s spheres of the indicated genotypes immunolabelled with α-catenin, ZO-1 and DAPI to show the loss of baso-lateral presence of α-catenin in the Tsc1 KO, with a concomitant appearance of an apical mislocalization relative to the apico-lateral marker ZO-1. Scale bar, 10 µm. d Representative pictures of pre-cystic post-natal day 20 mice, immunolabelled with Afadin antibody to show the localization of the protein in the indicated genotypes. Scale bar, 10 µm. e Representative pictures of pre-cystic post-natal day 20 mice, immunolabelled with α-catenin, ZO-1 and DAPI to show the localization of the proteins in the indicated genotypes. Source data are provided as a Source Data file.

Fig 5: Afadin controls activation of the thermogenic program in vivo. (a) l-Afadin, P-Afadin (S1795), Ucp1 and HSL protein levels in fat-specific Afadin knock-out (FAKO) and control littermates (CTRL). Representative blots are shown. (b) RT-qPCR analysis of Mllt4 mRNA levels in FAKO and control littermates. (c) Quantification of P-Afadin protein levels. (d) Rectal temperature measured every 30 min. (e) RT-qPCR analysis of Ucp1 mRNA levels in FAKO and control littermates. (f) Quantification of Ucp1 protein levels. N = 5–7. Data are presented as means + SEM, *p < 0.05 vs. CTRL at 5 °C, #p < 0.05 vs. 29 °C.