Cells were cultured in DMEM medium (low glucose) supplemented with 10% newborn calf serum at 37°C with 5% CO2. Cells were digested with 0.25% trypsin and subcultured at 70% to 80% confluence Exponentially growing A549 cells were used for all assays. Test compound Bostrycin (hydroxy-methoxy-tetrahydro-5-methyl anthracene dione), a novel compound isolated from marine fungi in P.R. China, was supplied by Marine Microorganism Laboratory, Institute of Chemistry and Chemical Engineering,

Sun Yat-Sen University. The chemical structure of bostrycin is shown inAdditional file 1, Figure S1. Major reagents Newborn calf serum, DMEM (low glucose), 0.25% trypsin digest, and Trizol reagent were purchased from GIBCO (Invitrogen Corporation, Carlsbad, CA, USA). MTT and DMSO were obtained from Sigma Corporation. Mouse anti-human phospho-Akt monoclonal antibody (mAb), rabbit anti-human DNA Damage inhibitor p110α mAb,

rabbit anti-human p27 mAb, horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (secondary antibody), HRP-conjugated goat anti-rabbit IgG (secondary antibody), see more and prestained protein molecular weight marker were purchased from Cell Signaling Technology (USA). Measurement of cell growth inhibition by MTT assay A549 cells were seeded in 96-well plates (5 × 103 cells per well) and treated with bostrycin (10, 20, and 30 μmol/L). Negative control wells (containing cells but not bostrycin), and the blank control (only medium) were plated with 6 replicates each. Untreated and treated cells were cultured at 37°C with 5% CO2 for 12 hours. MTT solution (20 μL) was added to each well and mixed; the wells were then incubated for an additional 4 hours. Culture supernatant was removed, DMSO (150 μL) was added to each well and vortexed at low speed for 10 minutes to fully dissolve

the blue crystals. Absorbance was measured at 570 nm (A570) and the percentage of growth inhibition of A549 cells was calculated at each time point and for each concentration of bostrycin according to the following formulae: % cell survival = (A570bostrycin group – A570blank)/(A570negative – A570blank) × 100% and % cell growth inhibition = 1 – % cell survival. Half maximal inhibitory concentration (IC50) values at respective Celecoxib times were then calculated using linear regression. Cell cycle and apoptosis rate assayed by flow cytometry A549 cells were cultured in 6-well plates (1.5 × 105 cells per well) and treated with different concentrations (5, 10, and 20 μmol/L) of bostrycin or complete DMEM medium (for the control group) and incubated for 24, 48 or 72 hours. Culture supernatant from each group was pooled and the cells were fixed for 12 h with 1 ml of 75% ethanol (106 cells/ml) and transferred to 2 mL Eppendorf tubes for flow cytometry and propidium iodide (PI) staining.