Remarkably, phase arrest declined after reaching a peak at

The identity of ATF was confirmed by Western blotting using poly clonal mouse anti ATF antibody, KU-55933 価格 Cell proliferation assay The effects of ATF, TPL or the combination on cell prolifer ation were assessed by the MTT assay. Cells in the exponen tial growth phase were seeded into a 96 well plate at a density of 5000 cells per well. After 24 h, ATF, TPL or the combination were added to the medium. The cells were incubated at 37 C for 24 h, then the cell via bility was determined by the colorimetric MTT assay at wave length 570 nm by TECAN Safire Fluores cence Absorbance and Luminescence Reader, The cell viability was calculated according to the for mula: Cell viability average A570 nm of treated group average A570 nm of control group × 100%. Each experiment was performed in quadruplicate and repeated at least three times.<br><br> To determine whether TPL in combination with ATF worked synergistically, the combination index in MTT assay was calculated as follows: CI AB, According to cell viability of each treatment, AB is the ratio of the combination treatment to the Linifanib 臨床試験 control treatment; A or B is the ratio of the single agent treatment to the control treatment. Thus a CI value less than, equal to or greater than 1 indicates that the drugs are synergistic, additive or antagonistic, respectively. A CI less than 0. 7 indicates that the drugs are significantly synergistic. Annexin V fluorescein isothiocyanate propidium iodide assay To quantify the percentage of cells undergoing apoptosis, we used the Annexin V FITC kit as described by the manu facturer.<br><br> Briefly, HCT116 and A549 cells were incubated for 24 hours purchase LY3009104 with TPL and ATF alone or in combination. Next, the treated cells were collected and trypsinized for 3 5 min. The digested cells were washed twice with cold PBS and resuspended in binding buffer at a concentration of 1 × 106 cells mL. After incubation, 100 uL of the solution was transferred to a 5 mL culture tube, and 5 uL of Annexin V FITC and 10 uL of PI were added. The tube was gently centrifuged and incubated for 15 min at room temperature in the dark. At the end of incubation, 400 uL of binding buffer was added, and the cells were analyzed immediately by flow cytometry, Flow cytometry analysis was performed with untreated HCT116 and A549 cells as control. Cell cycle analysis HCT116 cells were treated with TPL and ATF alone or in combination for 24 h.<br><br> Cells were then harvested, washed in PBS, resuspended gently in 5 mL of 100% ethanol, and fixed at 25 C for 1 h. After washing with PBS, cells were incubated with DNase free RNase A at 37 C for 1 h and washed with PBS. PI was added and the cells were incu bated at 37 C for 5 min. The distribution of cells with dif fering DNA content was analyzed on a FACSCalibur flow cytometer with CellQuest software at an excitation wavelength of 530 nm. Fluorescence emission was measured using a 620 nm band pass filter. Caspase activity assay Caspase 3 and caspase 9 activities were measured using colorimetric activity assay kits, The assay is based on the cleavage of the chromogenic substrates, DEVD pNA and LEHD pNA, by caspase 3 and caspase 9, respectively. Cells were lysed in chilled lysis buffer on ice for 10 min and centrifuged for 5 min at 10,000 × g.