3 methyladenine 3 ma

Cells were treated with ITF2357 and pemetrexed, either alone or in combination, as follows: (a) ITF2357; (b) pemetrexed; (c) ITF2357 and pemetrexed, simultaneously; (d) ITF2357 followed by pemetrexed; (e) pemetrexed followed by ITF2357. Both detached and adherent cells were collected, and differentially processed according to analyses performed.
3-Methyladenine (3MA, 1mM) (Enzo Life Science, Plymouth Meeting, PA, USA), and the pan-caspase inhibitor zVAD-fmk (zVAD, 50μM, Sigma-Aldrich) were dissolved in DMSO. Chloroquine diphosphate (CQ, 25μM, Sigma-Aldrich) was dissolved in water.

Shingosine kinase (SKi) (2-(p-hydroxyanilino)-4-(p-chlorophenyl)thiazole) was purchased by Calbiochem (EMD Chemicals, San Diego, CA, USA). Vincristine and doxorubicin were from Sigma-Aldrich (St. Louis, MO, USA). Bafilomycin A1, 3-methyladenine (3-MA), and Z-VAD-FMK were from Enzo Life Science (Plymouth Meeting, PA, USA). ROMe ((R)-FTY720 methyl ether) was synthesized according to Lim and colleagues [20 (link)]. Antibody to SK1 was as reported elsewhere [7 (link)]. For western blot analysis, primary antibodies were from Cell Signaling Technology (Danvers, MA, USA). For flow cytometric analysis, phycoerythrin (PE)-conjugated anti-cleaved caspase-3 (Asp175) was from Cell Signaling Technology. p-Akt and p-S6RP levels were performed as reported elsewhere [49 (link), 50 (link)].

Human XL Cytokine Array Kit (R&D Systems, Minneapolis, MN, USA) was used to compare cytokine profiles between TPE and transudate (T). Recombinant CXCL9 and CXCL10 and neutralizing antibodies against CXCL9 and CXCL10 were purchased from R&D Systems. The arginase inhibitor 2(S)-amino-6-boronohexanoic acid (ABH) was purchased from Cayman Chemicals (Ann Arbor, Michigan, USA) and used in cell and mouse experiments. The autophagy inhibitors 3-methyladenine (3-MA) and bafilomycin A1 (BafA1) were purchased from Enzo Life Sciences (Farmingdale, NY, USA).