Mct 4

Antibodies against MCT-1, MCT-4, FSP1, CAV-1, α-SMA, phospho-p38 (Thr180/Tyr182), p38, PAX6, STAT1, and total β-actin were obtained from Cell Signaling Technology. All other chemicals were purchased from Sigma-Aldrich. Recombinant human chemokines kits were purchased from R&D (Minneapolis, MN, USA).

Cells were seeded at a density of 5.0 × 10⁵ cells/well into 6 wells microplates (BD Biosciences, San Jose, CA) and incubated at 37°C and 5% CO₂. After 24 hours, cells were treated with 25 mM MDN-0066 and with DMSO as a negative control and incubated at 37°C for 24 or 48 hours. Cells were lysed in cell lysis buffer (Cell Signaling Technology, Danvers, MA). Twenty to fifty micrograms of protein (as determined by Pierce BCA Protein Assay Kit, Thermo Scientific) were resolved on 12% SDS-polyacrylamide gels and then transferred onto to a pre-wetted in methanol PVDF membrane that was blocked using SEA BLOCK Blocking Buffer (Thermo Scientific). Total HIF-1α, MCT4, C-Myc, Beclin, LC3b, LDHA, cleaved PARP (Cell Signaling Technology, Danvers, MA) and PKM2 (ProteinTech, Chicago, Illinois) were used as primary antibodies. β-actin (Sigma-Aldrich, St Louis, MO) was used as housekeeping protein for normalization. Anti-mouse and anti-rabbit DyLight 700 and 800 conjugated (Cell Signaling Technology, Danvers, MA) were used as secondary antibodies. Membrane was washed and read in Odyssey Infrared Imaging System (LI-COR Bioscience, Lincoln, Nebraska USA).

Compound 3K was obtained from SelleckChem (Houston, TX, USA). A solution of Compound 3K (15 mM) was prepared in DMSO. Culture medium and fetal bovine serum (FBS) were obtained from Gibco Invitrogen Corporation (Carlsbad, CA, USA). Horseradish peroxidase (HRP)-conjugated secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Primary antibodies against PKM2, PKM1, Cyclin B1, p-Cdc2, Cdc2, Bax, Bcl-2, caspase 3, Beclin-1, LC3B, Atg5, Atg7, p62, MMP-2, MMP-9, TIMP-2, p-AKT, AKT, p-AMPK, AMPK, p-mTOR, mTOR, p-p70S6K, p70S6K, GLUT1, LDHA, MCT4, and β-actin were all obtained from Cell Signaling (Beverly, MA, USA). MTT and DAPI were obtained from Thermo Fisher Scientific (Invitrogen, NY, USA).