Abt 199

WST-1 assay was performed according to the manufacturer's instructions (Roche, Mannheim, Germany) with 30 μl of WST-1 reagent was added to each well of a 24-well plate (1 × 10⁴ cell/well). After 1h of incubation using CO₂ incubator, the conversion of WST-1 reagent into chromogenic formazan was monitored with a spectrophotometer. On day 1 after plating, cells were treated with methyltransferase inhibitor, 5′-Aza-2′-deoxycytidine (Sigma, St. Louis MO, USA), 1 μM for 72 hr to induce demethylation of the cytosine residues, respectively and also added Bcl2 inhibitor, ABT-199 (Abmole BioScience), 1 μM for 24 hr. LPS (Sigma, St. Louis MO, USA) was applied to the gastric cancer cells at concentrations of 10 mg/ml.

Antibodies used for immunoblotting analyses in this study are listed below: rabbit anti-MCL-1 (1:500, ab32087), rabbit anti-BAX (1:1000, ab32503) were obtained from Abcam; mouse anti-BCL-2 (1:1000, #15071), rabbit anti-BIM (1:1000, #2933), rabbit anti-BCL-XL (1:1000, #2762), rabbit anti-BAK (1:1000, #12105), rabbit anti-AKT (1:1000, #4691), rabbit anti-p-AKT (1:1000, #4060), rabbit anti-mTOR (1:1000, #2983), rabbit anti-p-mTOR (1:1000, #5536), rabbit anti-p-4EBP1 (1:1000, #2855), rabbit anti-4EBP1 (1:1500, #9644), rabbit anti-FOXO3a (1:1000, #12829), rabbit anti-p-FOXO3a (1:1000, #9466) rabbit anti-cleaved PARP (1:1000, #5625) and rabbit anti-cleaved Caspase-3 (1:1000, #9664) were purchased from Cell Signaling Technology; mouse anti-β-actin (1:1000, sc-47778), mouse anti-GAPDH (1:1000, sc-32233) and rabbit anti-PUMA (1:1000, sc-28226) were from Santa Cruz Biotechnology; rabbit anti-p-4EBP1 (1:500, NB100-81769, used in Fig. 1b) and mouse anti-4EBP1 (1:1000, NBP1-47366, used in Fig. 1b) were obtained from Novus Biologicals. The compounds of BH3 mimetics (ABT263 and ABT199) and mTOR inhibitors (BEZ235, AZD8055, and Temsirolimus) were from AbMole Bioscience.