MTT Cell Viability Assay

Human pancreatic cancer cell lines (HPAC, PANC-1, HPAF-II, BxPC-3, and Capan-1), were seeded in 96-well plates at a density of 3000 cells per well. The next day, IL-6 (50ng/ml), INF-γ (50ng/ml), or Bazedoxifene (10μM) alone, or combination of IL-6 or INF-γ with Bazedoxifene were added in triplicate to the plates in the presence of 0% FBS in HPAF-II cells for 24 hours. Different concentrations of Bazedoxifene (5-10μM), paclitaxel (1-2.5 μM), or gemcitabine (5 μM) alone, or Bazedoxifene plus paclitaxel or gemcitabine were add in triplicate to the plates in the presence of 10% FBS in BxPC-3 or Capan-1 cells or paclitaxel plus gemcitabine were add in the plates in PANC-1, HPAC, BxPC-3 and Capan-1 cells. The cells were incubated at 37°C for a period of 24-48 hours. BxPC-3 and Capan-1 cells were seeded in 96-well plates at a density of 3000 cells per well and cultured at 37 °C. The next day, GP130 siRNA (100 nM) or negative control siRNA was transfected into cells in triplicate using lipofectamine 2000 for 72 hours. 25μl of 3-(4,5-Dimethylthiazolyl)-2,5-diphenyltetrazolium bromide (MTT, Sigma) was added to each sample in a volume of 100 μl and incubated for 4 hours. Then 150 μl of N, N-dimethylformamide (Sigma) solubilization solution was added to each well. The absorbance was read at 595 nm. Combination index (CI) was performed using data obtained from MTT assay with CompuSyn software. The CI values indicate a synergistic effect when <1, an antagonistic effect when >1, and an additive effect when equal to 1 (23).