Extended Data Fig. 6 |. LCN2 is a cold-induced mammokine involved in blocking cold-induced adipocyte UCP1 expression.

a and b) RNAScope FISH of indicated probes from mgWAT of RT or 24 h cold exposed (Cold) mice. Yellow asterisk indicate image used in Main Fig. 1f for Enho RNAScope. Scale bar at 50 μm. c) Real-time qPCR of indicated genes from mgWAT beige differentiated SVFs treated with 0–100 μm recombinant Adropin (Top) or 0–80 ng/ml recombinant LRG1 (Bottom) for 24 h. d) Real-time qPCR of indicated genes from mgWATs derived from 24 h cold-exposed WT or LRG1KO mice. N = 7,8. e) Gene correlation plots of Ucp1 and Lcn2 expression in WATs from male and female mice from the HMDP studies. f) Real-time qPCR of indicated genes from 24 h cold-exposed male and female iWATs. N = 6,6. g) tSNE plots of cell type clusters and normalized gene expression levels for Lcn2 across scRNA-seq datasets of female mgWAT SVFs and male iWAT SVFs. h) Real-time qPCR of Lcn2 in mgWAT EPCAM+ and EPCAM-cells from RT or 24 h cold-exposed mice. Inset shows violin plot of Lcn2 from scRNA-seq data. i) RNAscope FISH of indicated probes from mgWAT of 24 h cold exposed or Iso treated mice. Yellow asterisks indicate images used in Main Fig. 4b. Scale bar at 50 μm. j) Immunofluorescence microscopy for LCN2 or DAPI in mice exposed to 24 h cold or RT. Yellow outlines indicated ductal epithelium. Scale bar at 50 μm. k) The top five transcription factor regulons for luminal cell types at RT and COLD identified using SCENIC program. l) Subnetworks of transcription factor regulons of interest for Bhlhe41 in hormone sensing luminal cells (Up) and Elf5 in luminal alveolar cells (Down) derived using SCENIC. Transcription factors indicated in diamonds in the center and genes in the regulon in the surrounding circles. Genes in red were derived from the RT data, genes in blue were derived from the COLD data, and genes with both were present in the regulons in both conditions. Red arrow shows cold-induced mammokine Lcn2. m and n) Body weights (n) and scWAT weights (m) of 24 h cold exposed WT and Lcn2 KO male and female mice. N = 6,6 o) Real-time qPCR of indicated genes in organoids derived from K8rtTA-DTA mice treated with and without ISO and Doxycycline (Dox). Results from 3 organoid experiments. p-t) Confocal images of immunostaining of indicated antibodies in organoids derived from K8rtTA-DTA mice treated with and without Iso and Dox. Scale bar at 10 μm. Representative images from 3 organoid experiments. Each point represents three independent experiments (c, h, o). Unpaired two-tailed Student’s t test *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. Data are represented as mean ± S.E.M.