C57bl 6ncrl female mice

Female C57BL/6NCrl mice (N = 12, 8 weeks old; Charles River Laboratories, L’Arbresle, France) were used. At the start of the experiments, animals weighed (mean±SD) 22±2g. The mice were housed in groups of 6 and given five days to acclimate to the housing facility. Environmental conditions were a temperature of 22°C, humidity of 60%, lighting of 620 lux (6 bulbs of 20W) and a 12:12 light:dark cycle with lights on at 08:00 and off at 20:00. Animals were housed in 396x215x172 mm cages (1285L, Techniplast, France) filled with corncob (SAFE France) and given access to mouse maintenance food (A04, SAFE, France) and water ad libitum. Environmental enrichment included bedding (TopBrick and Topwoodwhool, SAFE France), coton and craft paper (SAFE France), one red tinted hut (SAFE France), one 10x10x50 mm aspen chew block (SAFE France). During housing, animals were monitored daily for health status. In an effort to reduce animal use in the long term, some healthy animals were scanned several times during the development and the optimization of the sequence. A completed ARRIVE Guidelines checklist is included in S1 ARRIVE Checklist.
As a mouse cancer model, female BALB/cByJ (N = 6, Charles River Laboratories, L’Arbresle, France) were injected with 10⁵ mouse renal carcinoma cells (RenCa) in 25μL under the renal subcapsule, as already described [26 (link)]. No adverse events were observed.

Female C57BL/6NCrl mice (n = 31), 6 weeks old upon arrival, were obtained from Charles River Laboratories (Lyon, France). All animals were group-housed (2–4 animals per cage) under controlled temperature (20–22 °C) and photoperiod (12:12 h light-dark cycle) and had unrestricted access to standard mouse chow and tap water. Mice were allowed to acclimatize to these conditions for a 1-week period prior to any experimentation. The experiment was replicated twice at different time points. Females were used according to the higher prevalence of functional and inflammatory gastrointestinal disorders in women [46 (link),47 (link)]. All procedures were approved by the Ethical Committee of the UniversitatAutònoma de Barcelona (protocols 1099 and 1101) and the Generalitat de Catalunya (protocols 5645 and 5646).

Female C57BL/6NCrl mice (n = 120) were purchased from Charles River (Wilmington, MA, USA) and allowed to acclimate to a low-fat control diet (10 kcal% from fat, Research Diets D12450J) ad libitum. At 36 weeks of age, mice were orthotopically injected into the fourth mammary fat pad with 50,000 metM-Wnt^lung mammary cancer cells in 50 μL sterile phosphate-buffered saline. Tumors grew until reaching a mean size of 100 mm³, at which time mice were block randomized to receive either vehicle, BMS-754807 (6.25 mg/kg), HCQ (60 mg/kg), CP (50 mg/kg), or combinations of two or three drugs. Doses used for in vivo analysis were based on the previous literature [31 (link),32 (link),33 (link),34 (link),35 (link),36 (link)]. BMS-754807 and/or HCQ were delivered by daily intraperitoneal injection in a vehicle of 30% PEG300, 5% DMSO, 5% Tween80, and 60% water. CP dissolved in water was delivered by once-weekly intraperitoneal injection. After 23 d of drug treatment (1–2 d prior to euthanasia), blood glucose was assessed via tail nick and portable glucometer (Bayer, Pittsburgh, PA, USA). Blood glucose assessment was performed 2 h and 6 h after drug treatment. Tumor sizes were monitored three times a week until average tumor size in the largest treatment group reached a volume of 1250 mm³, at which time all mice were euthanized, and tumors were excised and weighed.