Azd7545

Melanoma cell lines A375 (BRAF^V600E mutation), MeWo (BRAF/NRAS wild-type), SK-MEL-28 (BRAF^V600E mutation) and SK-MEL-2 (NRAS^Q61R mutation) were acquired from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultured as monolayers according to the supplier’s instructions in RPMI-1640 medium containing 11 mM glucose (PAN Biotech #17500, Aidenbach, Germany) with 3 mM L-glutamine (ThermoFisher Scientific #25030024, Waltham, MA, USA) and 10% FBS for a maximum of 50 passages. Sodium dichloroacetate (DCA) was purchased from Sigma-Aldrich (#347795, St. Louis, MO, USA) and AZD7545 from SelleckChem (#S7517, Houston, TX, USA). DCA was dissolved in either PBS or medium (1M stock concentration) and used in concentrations between 0–50 mM. AZD7545 was dissolved in DMSO (either 10 or 100 mM stock concentration), keeping the DMSO concentration at a maximum of 0.1%, and used in concentrations between 0–100 μM. The following antibodies were used: PDH (Cell Signaling, cat.nr. 3205, Danvers, MA, USA), pPDH^Ser293 (Calbiochem, AP1062, San Diego, CA, USA), pDPH^Ser232 (Calbiochem, AP1063, San Diego, CA, USA), pPDH^Ser300 (Calbiochem, AP1064, San Diego, CA, USA), PDK1 (Cell Signaling #3820, Danvers, MA, USA), PDK2 (Novus Biological, NBP2-75611, Englewood, CO, USA) and HSP90 (Santa Cruz, sc-13119, Dallas, TX, USA).

Selective BRAF inhibitors PLX4032 (Vemurafenib) and GSK2118436 (Dabrafenib) were purchased from Selleckchem. BRAF inhibitors were dissolved in DMSO according to the manufacturer’s instructions and stored at −80 °C. Working aliquots were diluted in 100% ethanol at a concentration of 1 mM for PLX4032 and 100 μM for GSK2118436 and stored at −20 °C. The MEK inhibitor GSK1120212 (Trametinib) was purchased from Selleckchem and was dissolved in DMSO at a concentration of 10 mM and stored at −20 °C. Dichloroacetic acid (DCA) was purchased from Sigma-Aldrich and dissolved in distilled water before use. AZD7545 was purchased from Selleckchem and was dissolved in 100% ethanol according to the manufacturer’s instructions. 10 mM working aliquots were stored at −20 °C. Mitoquinone mesylate (MitoQ) was purchased from Medkoo Biosciences, dissolved in DMSO according to the manufacturer’s instructions and stored at −20 °C. The following antibodies were used for western blot detection: anti phospho-PDH E1α (Merck Millipore); anti PDK1 (Enzo Life Sciences); anti phospho-ERK1/2 (Cell Signaling); anti ERK1/2 (Santa Cruz); anti cleaved-PARP (Cell Signaling); anti PARP (Cell Signaling); anti α-tubulin (Santa Cruz); anti HIF1α (Abcam); anti phospho- AMPK (Cell Signaling).

Cell lines were obtained from the American Tissue Collection Center (ATCC) and grown in high-glucose DMEM (HCT116, U2OS Hela, T47D) or RPMI1640 medium (H460, H1975) supplemented with 10% fetal bovine serum, 100 U ml⁻¹ penicillin and 100 μg ml⁻¹ streptomycin at 37 °C with 5% CO₂. All cell lines were authenticated by short tandem repeats profiling and tested negative for mycoplasma contamination. Murine Kras^G12D/wt;Trp53^Δ/Δ lung adenocarcinoma cells from Adeno-Cre infected Kras^LSLG12D/wt;Trp53^flox/flox mice^{33 (link)} were cultured in RPMI1640 medium supplemented as above. CDDP^naïve mouse tumor cells were derived from untreated tumors, CDDP^res cells from tumors that had relapsed after an initial CDDP treatment. Chemotherapy drugs and inhibitors were obtained from Sigma-Aldrich unless indicated otherwise and used at the following concentrations: CDDP 1 µg ml⁻¹, oxaliplatin 5 µg ml⁻¹, carboplatin 5 µg ml⁻¹, etoposide 1 µm, doxorubicin 0.04 µg ml⁻¹, RITA (Merck) 1 µm, DCA 20–40 mm, 2DG 10–30 mm, metformin 1.25–4 mm, phenformin 60–125 µm, AZD8055 (Selleckchem) 0.2–1 µm, Rapamycin (Selleckchem) 250 nm, Everolimus (Absource Diagnostic) 250 nm, AZD7545 (Selleckchem) 10 µm, chloroquine 25–100 µm, 3-methyladenine (Calbiochem) 5 mm, and SBI-0206965 (Biovision) 5 µm. For starvation experiments, cells were grown in Hank’s Balanced Salt Solution (Sigma) for up to 5 days.