B6 cg lepob ob ob

To control for the effects of adipose tissue content in mice, 4 week-old female B6.Cg-Lep^ob (ob/ob; Jackson Laboratory) mice were purchased and maintained on normal rodent diet. These mice gain weight due to a homozygous mutation in the leptin (Lep) gene that causes excessive eating and rapid weight gain. Weight was monitored over time beginning at 5 weeks old, and mice were euthanized when they reached >40g. This time period was significantly shorter (6 weeks) than that of the DIO model (15 weeks). After 6 weeks, animals were euthanized for flow cytometry analysis of myeloid cell populations in the lung, or injected with tumour cells for 48 h metastasis assays (see ‘Experimental metastasis assay’ section).

Animal experiments were in compliance with the ethical regulations set by UC Berkeley Animal Care and Use Committee. Male C57BL/6 (wild type) mice (Jackson) or B6 Cg-Lep^ob (ob/ob) (Jackson) were used at 8 wks of age. Neither randomization nor blinding was employed. A sample size of 5 per group was initially used to assess whether differences could be detected and was found to be sufficient to achieve statistical significance. For fasting and refeeding experiments, mice were fasted overnight and then fed a high carbohydrate, fat free diet for 6 hrs. For knockdown or overexpression experiments, mice received through tail vein injection 100μl of adenovirus (MED17 shRNA, MED17 WT, MED17 S53A, or CK2α1 shRNA; Vector Biolabs) at 2.0 × 10¹⁰ PFU/mL. Twelve days after injection mice were fasted overnight then refed high carbohydrate diet for 6 hrs. Knockdown or overexpression was verified by measuring mRNA or protein abundance prior to qPCR analysis of lipogenic markers. For CK2 inhibition experiments, mice were injected with either vehicle or CX-4945 (75mg/kg; APEX Bio) intraperitoneally for 5 days, then fasted overnight and refed high carbohydrate diet for 6 hrs.