B6.129 leprtm2 cre rck j

We used the LepR-reporter mouse to visualize LepR-expressing cells. LepR-reporter mice were generated by breeding the LepRb-IRES-Cre mouse (B6.129-Leprtm2(cre)Rck/J, Jackson Laboratories) with the Cre-inducible tdTomato-reporter mouse (B6;129S6-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/J, Jackson Laboratories). This mouse model has been validated6 (link)45 (link). Only cells that express the long form of LepR contain the red fluorescent tdTomato protein in the LepR-reporter mouse. To identify the localization of LepR-expressing cells in the mammary tissue, the inguinal glands of four pregnant LepR-reporter mice were dissected at specific developmental time-points (16–18 days of pregnancy). The tissue was fixed in 4% paraformaldehyde overnight at 4 °C and processed for hematoxylin-eosin staining or immunostaining according to the following procedures: 4-μm sections were deparaffinized, rehydrated, and subjected to 10 mM citrate buffer pH 6,0 antigen retrieval for 30 min at 95 °C. Anti-tdTomato (Clontech) or Anti-CK5 (Abcam) primary antibodies were incubated overnight at 4 °C at 1:200 dilution, and Alexa fluor 488-conjugated and Alexa fluor 633-conjugated secondary antibodies were used, respectively, at 1:750 dilutions. Samples were mounted in Prolong Gold antifade reagent with DAPI (Molecular Probes).

The deletion of Socs3 gene in LepR‐expressing cells was achieved by breeding the LepR‐IRES‐Cre strain (B6.129‐Lepr^tm2(cre)Rck/J, Jackson Laboratories) with the SOCS3‐floxed mouse (B6;129S4‐SOCS3^tm1Ayos/J, Jackson Laboratories), as previously described and validated (Pedroso et al. 2014, 2016; Zampieri et al. 2015, 2016; Bohlen et al. 2016). Animals carrying the SOCS3 conditional deletion in LepR‐expressing cells (SOCS3 KO group) were homozygous for the loxP‐flanked Socs3 and LepR‐Cre alleles. The control group was composed of littermate animals carrying only the LepR‐Cre allele in homozygosity. Control and SOCS3 KO mice were weaned at 3–4 weeks of age and their mutations were confirmed by genotyping the DNA that had been previously extracted from the tail tip (REDExtract‐N‐Amp™ Tissue PCR Kit, Sigma). Mice were maintained under standard conditions of light (12‐h light/dark cycle) and temperature (23 ± 1°C). All animal procedures were approved by the Ethics Committee on the Use of Animals of the Institute of Biomedical Sciences at the University of São Paulo, and were performed according to the ethical guidelines adopted by the Brazilian College of Animal Experimentation.

All experiments were carried out in compliance with the Association for Assessment of Laboratory Animal Care policies and approved by the University of Virginia Animal Care and Use Committee (Figs. 3 to 10 and figs. S5 to S13). Animals were housed on a 12:12-hour LD cycle with food (PicoLab Rodent Diet 5053) and water ad libitum unless otherwise indicated. All experiments were performed on male mice 12 weeks or older unless otherwise indicated. Wild-type C57BL6/J mice, LepR-Cre [B6.129-Lepr^tm2(cre)Rck/J, The Jackson Laboratory, #008320, RRID:IMSR_JAX:008320] (100 (link)), Ai14 tdTomato reporter line [B6.Cg-Gt(ROSA)26Sor^{tm14(CAG-tdTomato)Hze}/J, strain #007914, RRID:IMSR_JAX:007914] (101 (link)), and Pdyn-Cre [B6;129S-Pdyn^{tm1.1(cre)Mjkr}/LowlJ, The Jackson Laboratory, #027958, RRID:IMSR_JAX:027958] (113 (link)) mice were used.