Fig. 2.
SH-130 enhances radiation-induced apoptosis. DU-145 cells were seeded into 6-well plates at the concentration of 2×105/ml, pre-treated with zVAD (2.5 μM) for 1 h, incubated with 10 μM of SH-130 or SH-123, and irradiated at doses of 0, 20 or 30 Gy, respectively. Twenty-four hours after incubation, cells were harvested and processed for further detection. Data represented one of three independent experiments. A, early apoptotic cell populations after treatment. DU-145 cells were stained with Annexin V-FITC and PI (Trevigen) and determined by flow cytometry. Data represented the mean ± SD (n=3), and were analyzed by Student’s t test. ** P<0.01, *** P<0.001. B, Western blot analysis of apoptosis-related proteins. Samples were probed with antibodies against PARP, caspase 3, XIAP, and Smac. Actin is shown as a loading control. Band intensity of cleaved PARP was normalized to Actin. C, caspase 3 activation after treatment. Whole cell lysates (20 μg) were reacted with fluorogenic substrate DEVD-AFC. After incubation at 37 °C for 2 h, released AFC was monitored by a microplate reader (BMG LABTECH). Data were shown as mean ± SD (n=3). Fold increase of fluorescence signal was expressed by normalizing activity to untreated control.