Extended Data Fig. 3: Reduced energy expenditure and thermogenesis in IL-27 signalling deficient mice.

a–c. IL-27Rα KO and WT mice at 8 weeks of age were placed in metabolic cages. The food intake (a, n = 8 for WT and n = 7 for KO), oxgen consumption (b, n = 8) and energy expenditure (c, n = 8) were shown. d. BAT and SCW tissues were isolated from WT-ND and IL-27Rα KO-ND mice at 6-8 weeks of age, cut into small pieces (~0.003g for BAT and ~0.004g for SCW) and used for detection of basal oxygen consumption rate by Seahorse XF Analyzer (n = 5 mice per group, 18 pieces for WT-BAT, 23 pieces for KO-BAT and 17 pieces for SCW samples). e & f. IL-27Rα KO and WT mice at 8 weeks of age were fed on HFD for 10 weeks, SCW were collected for RNA-seq analysis. e. Gene set enrichment analysis was performed for indicated pathways. Genes were ranked according to their expression. NES, normalized enrichment score; FDR, false discovery rate. f. Heat maps of differentially expressed genes in indicated pathway. g. Gene expression in SCW from IL-27Rα KO and WT mice on normal diet were determined by real-time PCR (n = 9 for WT-Ucp1, WT-Cidea, WT-Adipoq and WT-Retn, n = 10 for WT-Ppargc1a, WT-Ppara, WT-Cox8b, WT-Cox5a, WT-Prdm16 and WT-Elovl3, n = 11 for WT-Pparg, WT-Fabp4 and WT-Lep, n = 8 for KO-Ucp1, n = 10 for KO-Retn, n=11 for the rest KO mRNAs). h. Survival curve of IL-27Rα KO and WT mice fed on ND in response to cold challenge (4 °C, n = 9 for WT and n=11 for KO). i. Rectal temperature of mice fed on ND in response to cold challenge (4 °C, n = 9 for WT and n = 11 for KO). j–l. EBI-3 KO mice and WT controls fed on normal chow were challenged at 4 °C. The survival curve (j) and rectal temperature (k) were recorded and shown (n = 5 for WT and n=6 for KO). After 12 hours of cold stimulation, BAT and SCW were collected, lysed and used for immunoblotting analysis of UCP1 (l). m-p. EBI-3 KO mice were i.p. injected with rmIL-27 (100µg/kg) or PBS every day for 7 days and then challenged at 4°C. m. Rectal temperature of mice in response to cold was shown (n = 6). n. Immunoblot analysis of protein extracts from SCW and BAT after 24 hours of cold challenge. UCP1 staining (o) and histology analysis (p) (Scale bar = 100µm). Representative sections were shown. (l & n) Each lane represents one biological independent sample and band densities were quantified with ImageJ, ratios of UCP1/HSP90 were normalized. All experiments were repeated at least twice with similar results. Data are mean ± s.e.m. Data represent biologically independent samples except for d. Two-tailed unpaired student’s t-test (d & g); two-way ANOVA (a–c); two-way ANOVA with Sidak’s multiple comparisons test (i, k & m); log-rank test (h & j); Gene Set Enrichment Analysis (e).

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