Atorvastatin

Tumour-associated IMCs were freshly isolated from Braf^{+/LSL−V600E};CreER^+/0 mice and cultured in serum-free Dulbecco's modified Eagle medium (DMEM) (Invitrogen) as previously described (Kamata et al., 2015 (link), 2020 (link)). Atorvastatin (3.3 µM, Generon), lonafarnib (1-5 µM, Tocris Bioscience), GGTI-298 (8 µM, Tocris Bioscience), ML141 (10 µM, Merck) and/or EHT1864 (1-10 µM, Tocris Bioscience) were added to the serum-free IMC culture for 72 h. IMCs cultured for 96 h in serum-free DMEM were treated with the CCR1 inhibitor J113863 (5 µM, Tocris Bioscience) for 1-24 h as indicated. For membrane protein purification and detergent-insoluble protein analysis, primary IMCs were cultured for 48-72 h in DMEM containing 1% foetal bovine serum (Invitrogen) supplemented with Atorvastatin (3.3 µM), epoxomicin (0.05 µM, Sigma-Aldrich) and/or MG132 (3.3 µM, Sigma-Aldrich).

All animal experiments were performed under UK Home Office Licence authority. Braf^{+/LSL−V600E}, Kras^+/LSL−G12D and CCAGCreER^TM mice were backcrossed onto the C57BL/6J strain and genotyped as reported (Kamata et al., 2017 (link), 2020 (link)). Male and female animals >8 weeks of age were used for experimental procedures. Nasal delivery of Ad5-CMV-Cre or Ad5-mSPC-Cre was performed as described (Kamata et al., 2017 (link), 2020 (link)). Atorvastatin (10 mg/kg, Generon) was orally administered once daily for the indicated periods. Lung tissues were processed for Haematoxylin and Eosin (H&E) staining and immunohistochemistry (IHC) as described (Kamata et al., 2020 (link)).

Thirteen-week-old female VillinCre^ER/0/Braf^V600E-LSL/+ mice received either 10 mg/kg atorvastatin by oral gavage or 5% DMSO/PBS vehicle control daily for 12 days. A 20 mg/mL atorvastatin (Generon) stock in DMSO was freshly diluted to a 1 mg/mL working solution in PBS. Days 1–7 of the study were a daily pre-treatment period of atorvastatin/vehicle. Days 8–10 consisted of both atorvastatin/vehicle gavage and intraperitoneal tamoxifen injection to induce Braf^V600E expression. On the final day of the study at 3 days post Braf^V600E induction, mice received intraperitoneal injections of 200 μL of 20 mM BrdU solution (Sigma) 3 h before tissue harvesting.