The cells were then incubated with chemotherapeutic agents in serum free medium for additional

24 hr (Doxo) or 48 hr (5-FU and Gem), since it was the optimal incubation time for each drug. NQO1 enzyme activity assay NQO1 Thiazovivin assay was performed according to the method described previously [20]. Cells were seeded at 7.5 × 103 cells/well in flat-bottomed 96-well cultured plates overnight. After cells were cultured for the designated time, cells were lysed with 50 μL solution containing 0.8% digitonin and agitated on a shaker at room temperature for 10 min. Twenty-five microliter of 0.55% dicoumarol was added into culture wells designated as baseline activity, while the corresponding paired wells were added with distilled water (DW) designated as the test activity wells. After that, all wells were added with 200 μL of reaction mixture (the following

stock solution was prepared for each set of assay: 7.5 mL of 0.5 M Tris–HCl (pH 7.4), 100 mg of bovine serum albumin (BSA), 1 mL of 1.5% Tween-20 solution, 0.1 mL of 7.5 mM FAD, 1 mL of 150 mM glucose-6-phosphate, 100 μL of Pinometostat supplier 50 mM β-NADP, 275 unit of yeast glucose-6-phosphate dehydrogenase, 45 mg of MTT, and DW to a final volume of 150 mL and menadione (1 μL of 50 mM menadione dissolved in acetonitrile per milliliter of reaction mixture) was added just before the mixture is dispensed into the microtiter plates. A blue color developed and the plates were placed into a microplate reader with filter wavelength of 620 nm and readings were made at 0.5 min interval for about 10 min. The rate of increase of the optical readings with times represents the activity of the reaction. Using the extinction coefficient of MTT formazan of 11,300 M-1 cm-1 at 610 nm and correction for the light

path of the microplate, NQO1 activity was expressed as nmol/min/mg protein. Cytotoxicity or SRB assay Cytotoxicity testing is used to evaluate the effects of chemotherapeutic agents. In brief, CCA cells were seeded onto 96-well cultured plates at a density of 7.5 × 103 cells/well overnight, then media was renewed with fresh media Thymidine kinase containing test Hedgehog antagonist compound and further incubated for the indicated times. Assay was performed at the endpoint of treatment to determine amount of protein remaining in each well. Media was discarded and replaced with 100 μL of ice-cold 10% trichloroacetic acid (TCA) and placed in 4°C for at least 1 hr. Then TCA was removed and wells were carefully rinsed with deionized (DI) water for 5 times. After 10 min of air drying, 50 μL of 0.4% sulforhodamine B (SRB) in 1% acetic acid was added for 30 min. Cells were rinsed 3–4 times with 1% acetic acid and air dried for 1 hr at room temperature. Finally, adhered cells were solubilized with 200 μL of 10 mM Tris base and plates were shaken for 20 min before absorbance reading with a microplate reader with filter wavelength of 540 nm.