Western blotting HSP70, HSP90, actin, tyrosine phosphorylated and unphosphorylated STAT3 were quantitated by western blot examination as previously described.The antibody against HSP70 was obtained from R&D Systems, Minneapolis, MN, and the antibody towards HSP90 was purchased from Santa Cruz Biotechnology, Santa Cruz, CA.The antibody towards P-STAT3 was obtained from Upstate Biotechnology, Lake Placid, NY.To detect the unphosphorylated protein, the immunoblots mTOR signaling pathway had been reacted with an antibody towards the NH2 termini of STAT3.The immune complexes were visualized by the enhanced chemiluminescence reaction.All experiments were conducted at least in triplicate unless otherwise stated.Initially , both total STAT3 and actin were used to normalize for PSTAT3 but because the results have been similar, actin was used as a housekeeping gene in the current study.Apoptosis measurement Apoptosis of cells was evaluated by double staining with fluoresceine-isothiocyanate -labeled annexinV and 7-Aminoactinomycin D.Briefly, 2 ? 104 cells were washed twice in cold PBS and had been resuspended in 0.25 ml of binding buffer.Five microliters of each FITC-annexin V and 7AAD had been added to the cells, and the mixtures were gently vortexed and incubated for 15 min at room temperature in the dark.
Within 1 h, the cells were analyzed at 488 nm using FACSCaliber flow cytometer.Interaction assays All assays had been conducted at least in triplicates as previously described.The Hill function was fitted to each concentration?response curve for each drug.
After fitting and determination of the IC50, five combination ratios of the IC50 were characterized.Pharmacodynamic drug?drug interaction model Interaction of ATO and 17-DMAG on the inhibition screening compounds of P-STAT3 had been characterized with the following equation for non-competitive interaction.Symbol A refers to the concentration of ATO and B refers to 17-DMAG and Imax is the fraction which represents the maximal capacity by which drug A or B can inhibit constitutive STAT3 activity when present alone.When Imax = 0, it indicates no possible inhibition and when Imax = 1, it indicates complete inhibition of response at high concentrations.The IC50 is the concentration of drug A or B alone which elicits half the maximal response and ? is a power or curve shape coefficient.The interaction of ATO and 17-DMAG on the stimulation of HSP70 expression was characterized with the following stimulatory equation for non-competitive interaction.Symbol A refers to the concentration of ATO, B refers to 17-DMAG, Smax is the maximum capacity of either drug on the stimulation of HSP70 when present alone and SC50 is the concentration which produces half the maximum effect when the drugs are present alone.In the above equations, the values of Imax vary between 0 and 1, but the values of Smax are greater than zero with no upper limit.