Ml141

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).

Migration assays were performed in 96-well chemotactic chamber with 10 μm pore at a density of 10³ pores per mm² (Neuro Probe Inc, Gaithersburg, USA). Cells were pre-treated with Mitomycin C (16 h, 30 μM) (Sigma®), an alkylating agent used to generate mitotically inactive cells in culture. Other treatments were administrated as follows: Alisertib (48 h, 5 nM; Selleckchem®), AMD3100 (24 h, 25 nM; Sigma®), U0126 (24 h, 10 μM; Cell Signaling Technology®), ML141 (24 h, 1 μM; Merck Millipore®) (n = 3). Migration assays were performed as previously described [9 (link)].
Briefly, cells were stained with Cell Tracker Green (CTG) (30 min, 5 μM, 37 °C; ThermoFisher®) in pre-warmed serum-free DMEM and washed in fresh serum-free DMEM during 30 min at 37 °C. Twenty-five thousand cells were seeded in the upper wells of the chemotactic chambers. The lower chamber was filed by 30 μl of serum-free DMEM supplemented with 500 nM CXCL12 (Peprotech®) or control solution. After 16-h incubation in a 5% CO₂ humidified incubator at 37 °C, cells having migrated in the lower chambers were fixed with 4% paraformaldehyde (PFA) for 15 min and rinsed 3 times with deionized water. The percentage of migrating cells was quantified by counting the number of CTG-positive cells per area by fluorescent microscopy using the Image J software.

The mTORC2 activator A-443654 (MCE) was made up to a 40 mM stock in DMSO, and 20 μL of 8 mM dilution made in 4% PEG, 4% DMSO in 0.9% saline was injected beneath the skin of the left hind paw (intraplantar). The Rac1 inhibitor NSC23766 (EMD Millipore) was prepared in a 50 mM stock solution in distilled water, and 10 μL of 10 mM dilution in 0.9% saline was injected beneath the skin of the left hind paw. The Cdc42 inhibitor ML141 (EMD Millipore) was made up to a 100 μM stock in DMSO, and 10 μL of 50 μM dilution in 0.9% saline was injected beneath the skin of the left hind paw. The rapalog CCI-779 (Sigma-Aldrich) was made up to a 50 mg/mL stock in DMSO and was then diluted to a dose of 25 mg/kg in 5% PEG, 5% Tween-80 in saline.

MEG-01 cells (3 × 10⁵ cells/ml) were seeded in 6-well plates and incubated with medium containing 2 mM valproic acid (VPA, FUJIFILM Wako Pure Chemical) for up to 12 days with medium containing 2 mM VPA added on the 7th day. Cdc42 inhibitors ML141 (Merck Millipore), R-ketorolac (Sigma-Aldrich) and geranylgeranyl transferase inhibitor GGTI-298 (CAYMAN CHEMICAL, Ann Arbor, MI) were added once every 3–4 days. After differentiation, attached cells were harvested by scraping and combined with floating cells. Collected cells were spin down at 70xg for 10 min and the supernatants were further centrifuged at 1,500xg for 10 min to harvest PLP. Differentiated cells or PLP were fixed in 2% paraformaldehyde (PFA)/PBS for 30 min at 4 °C and then washed with PBS. Pellets were suspended in 0.3 ml of PBS containing sodium azide and stored at 4 °C for analysis by flow cytometry.