Sbi 0206965

Stock solutions of reagents were prepared by dissolving the reagents in dimethyl sulfoxide (DMSO, Sigma-Aldrich). SBI-0206965, GSK 2606414, and MRT 68921 were purchased from Selleck Chemicals (Houston, TX, USA). Bafilomycin A1 and 3-methyladenine (3-MA) were purchased from Sigma-Aldrich. Hydroxychloroquine (HCQ) was purchased from Myung-in Pharmaceuticals. z-VAD-FMK was obtained from R&D Systems. Control cells were treated with equal amounts of DMSO.

The human cancer cell lines A549, H1299, NCI-H460, MNK45, U251, SW480, SW620, HCT116, Colo320 and HT-29, PC-3, U266, and the mouse breast cancer cell line 4T1 were cryopreserved in the Hematological Laboratory of Zhujiang Hospital (Guangzhou, China). All cell lines were incubated in DMEM medium supplemented with 10% fetal bovine serum at 37 °C with 5% CO₂. WZ4003, SBI-0206965, Chloroquine, and MRT68921 were purchased from Selleckchem (Houston, TX, USA), dissolved in DMSO or water, and stored at −20 °C. CCK-8 was purchased from Dojindo Laboratories (Japan). Mitotracker, DAPI, and TritonX-100 were purchased from Solarbio (Beijing, China).

Cells were serum-starved overnight in a serum-free medium and treated with PBS or stimulated for the indicated time points with 100 ng/mL of FGF1, FGF7 or FGF10¹³. Where indicated, cells were pre-incubated for 2 h with DMSO or 100 nM PD173074 (Selleckchem, #S1264), 0.5 μM Rapamycin (Sigma-Aldrich, #37094), 2 μM ULK101 (Selleckchem, #S8793), 10 μM SBI0206965 (Selleckchem, #S7885), 10 μM Dynasore (Abcam, #ab120192), Monensin sodium salt, Na⁺ ionophore (Abcam, #ab120499) and 200 μM Primaquine bisphosphate (Sigma-Aldrich, #160393). Control cells were pre-incubated with DMSO alone. Cells treated with glucose-6-phosphate (Sigma-Aldrich, #10127647001), Sodium valproate (Sigma-Aldrich, #BP452) and Fluspiriline (Sigma-Aldrich, #F100) were treated for 24 h prior to FGF10 stimulation. After stimulation, cell extraction and immunoblotting were performed. Proteins were resolved by SDS-PAGE and transferred to nitrocellulose membranes (Protran, Biosciences). Proteins of interest were visualised using specific antibodies, followed by peroxidase-conjugated secondary antibodies and by an enhanced chemiluminescence kit (Amersham Biosciences). Blots were visualised using the Universal Hood II Gel Molecular Imaging System (Bio-Rad). Each experiment was repeated at least three times and produced similar results.

LPS from Escherichia coli 0111:B4, oligomycin, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), rotenone, mitomycin C, HMGB1, and BrdU were purchased from Sigma-Aldrich. The following kinase inhibitors were purchased from Tocris: leucine-rich repeat kinase 2-IN-1, BMS509744, GSK650394, and GSK2250665A. IRAK-1/4 inhibitor 1 was purchased from Sigma-Aldrich, SBI-0206965 and GNF-2 were obtained from Selleckchem, P-M2tide was obtained from Enzo Life Sciences, and MlkL inhibitor was obtained from Calbiochem. AZD7545 was kindly provided by Professor In-Kyu Lee at Kyungpook National University.

SBI‐0206965 was from Selleck (Houston, TX, USA), and MRT68921 was from Sigma (St. Louis, MS, USA). ULK1 antibody (#8054), LC3B antibody (#3868), the cell‐cycle regulation antibody sampler kit II (#9870) and the horseradish peroxidase‐linked anti‐rabbit and anti‐mouse IgG were all from Cell Signaling Technology (Danvers, MA, USA). The anti‐β‐Tubulin, anti‐(glyceraldehyde 3‐phosphate dehydrogenase) and anti‐β‐actin Ig and TransStart FastPfu DNA Polymerase were from Beijing TransGen Biotech (Beijing, China). Nocodazole was from Selleck. Propidium iodide (PI)/RNase staining buffer was from BD Pharmingen (San Diego, CA, USA). The siRNAs were ordered from Genepharm (Shanghai, China), and primers were from Sangon Biotech (Shanghai, China). HiPerFect was from Qiagen (Dusseldorf, Germany). Protease inhibitor and phosphatase inhibitor cocktails were from Roche (Basel, Switzerland), and the polyvinylidene fluoride membrane was from Millipore (Billerica, NJ, USA). GlutaMAX was from Gibco (Carlsbad, CA, USA). The secondary fluorescently conjugated antibodies, Antifade ProLong Gold, were from Molecular Probes (Eugene, OR, USA). M‐MLV Reverse Transcriptase and TRIzol were from Invitrogen (Waltham, MA, USA). Prestained Protein Ladder (26616) and M‐PER were from Thermo Pierce (Waltham, MA, USA). CellTiter‐Glo (#G7570) was from Promega (San Luis Obispo, CA, USA).

Original samples from the PKIS were obtained from GSK. The compounds GW301784X, GW837331X, GW406108X, GSK2358994A, GSK846226A and GW429374A were synthesised according to previously published methods [20–25 (link)]. Protein kinases (ULK1, VPS34 and AMPK α1β2ɣ1) and SAMS peptide were purchased from MRC PPU Reagents and Services, Dundee, Scotland. ADP Hunter™ Plus kits and ultra-pure ATP Gold were from Discoverx. SBI-0206965 and VPS34-IN1 were obtained from Selleck Chemicals. MRT68921 was provided by LifeArc (formerly MRC-Technology). Trizma® hydrochloride, Trizma® base, Tris(2-carboxyethyl)phosphine (TCEP), Pluronic® F-127, sodium chloride (NaCl), magnesium chloride (MgCl₂), manganese chloride (MnCl₂), ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), dimethyl sulfoxide (DMSO) and bovine myelin basic protein (MBP) substrate were from Sigma–Aldrich. 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), Tween-20 and PI:PS lipid kinase substrate were from Thermo Fisher Scientific. Reagents used for Western blot and immunofluorescence were published previously [26 (link)].