Eht1864

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).

Losartan, lisinopril and AngII were purchased from Wako Pure Chemical (Tokyo, Japan). Benazepril was purchase from Tokyo Chemical Industry (Tokyo, Japan). PD123319, ZD7155, A779, SR202 and EHT1864 were purchased from Tocris Bioscience (Bristol, UK). Diphenylene iodium (DPI) and 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF) were purchased from Sigma-Aldrich. A water-soluble derivative of forskolin, 7-deacetyl-7-[O-(N-methylpiperazino)-γ-butyryl]-forskolin (forskolin-ws), was purchased from Calbiochem (San Diego, CA, USA).
Antagonists of AT1R (losartan and ZD7155) and AT2R (PD123319), the Mas receptor (A779) and PPARγ (SR202) and inhibitors of ACE (benazepril and lisinopril), NOX (DPI and AEBSF) and Rac (EHT1864) were dissolved in the perfused solution and administered to the striatum through the probe during the experimental period. Forskolin-ws, which stimulates cAMP production by activating adenylyl cyclase^{48 (link)}, and AngII were dissolved in sterilized physiological saline (Otsuka Pharmaceutical, Tokyo, Japan) and directly administered into the striatum through the thin needle of the MI-A-I-8-03 probe using an ESP-32 pump. The flow rate was 0.1 μL/min, and the total volume was 1 μL for forskolin-ws and 2 μL for AngII.

For differentiation assays, EpH4 cells were cultured in DMEM-F12 supplemented with 5 µg/ml insulin, 1  μg/ml hydrocortisone and antibiotics overnight with 2% Matrigel (LrBM, BD Biosciences) added to the culture medium (LrBM overlay) as required. Cells were stimulated with 3 µg/ml sterile-filtered sheep pituitary Prl (Sigma #L6520; 150 nM final Prl concentration) for 15 minutes. EpH4 cells were treated with inhibitors (Pitstop-2 [15 µM, Abcam]; Dyngo-4a [60 µM, Abcam]; Filipin III [8 µM, Sigma-Aldrich]; AZD-1480 [10 µM, Santa Cruz Biotechnology]; EHT 1864 [25–100 µM, Tocris]) or an equal volume of DMSO (where appropriate) diluted in differentiation medium for 15 minutes at 37 °C before addition of Prl.

Abs against P-Axl(Y702) (rabbit), P-AKT(S473) (rabbit), anti-Mer (mouse), P-src (Y416), and anti-P-Tyrosine (P-Tyr-100) (mouse) were obtained from Cell Signaling Technology (New England BioLabs, Beverly, MA, USA); anti-Axl (goat), anti-FAK (rabbit), anti-Tyro-3 (goat), and anti-β1 and anti-p130Cas (mouse) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-α-tubulin (mouse) from Thermo Scientific (Fremont, CA, USA); anti-β-actin (rabbit) from Sigma-Aldrich (Saint Louis, MO, USA); anti-β3 from Abcam (Cambridge, UK); and anti-β4 from Millipore (Merck Millipore, Oxford, UK).
Alexa Fluor 488 phalloidin was obtained from Molecular Probes (Invitrogen, Carlsbad, CA, USA). Human recombinant Gas6 and Axl-Fc were from R&D systems, Inc. (Minneapolis, MN, USA), as was the ELISA for Gas6 dosage. FN was from Sigma-Aldrich. The Taqman Gene Expression Assays were from Applied Biosystems (Foster City, CA USA). Puromicine and Lipofectamine 2000 were from Invitrogen. R428, the Axl inhibitor, was from Rigel (South San Francisco, CA, USA); EHT1864, the Rac inhibitor, was from Tocris (Minneapolis, MN, USA).