Bl6 mice

To model obesity with diet, 5-week-old female BL6 mice (Jackson Laboratory) were enrolled on either high-fat (HF; 60% kcal, Research Diets D12492) or low-fat (LF; 10% kcal, Research Diets D12450) irradiated rodent diet for 15 weeks. Weight was monitored over time. After 15 weeks, animals were either euthanized for flow cytometry, or injected with tumour cells (see ‘Primary tumour growth assay’ and ‘Experimental metastasis assay’ sections). For the diet-switch model (HF–LF), 5-week-old female BL6 mice were fed for 15 weeks with HF diet, and then switched to LF diet for 7 weeks⁶⁴ prior to euthanization.

Female C3H/HeJ (C3H) mice were bred to male BL/6 mice (both strains have wild-type Zhx2 alleles; The Jackson Laboratory), and female mice were monitored for vaginal plugs to estimate the time of fertilization. For embryonic day 17.5 (e17.5), pregnant females were euthanized by CO₂ asphyxiation at 17.5 days postconception, and fetuses were removed. For postnatal time points, mice were killed at the designated times after birth. The e17.5 fetuses and postnatal day 7 (p7) mice were euthanized by decapitation; mice at later postnatal time points were euthanized by CO₂ asphyxiation. Livers from mice at all time points were isolated, frozen, and stored at −80°C.