Fig. 6
NCTD induces apoptosis of HT-29 cells, HDLECs and the co-culture system in vitro. a Hoechst and CY3 immunofluorescent dyes (fluorescent microscope, magnification × 200). No apoptotic cells were observed in control group (0 μg/ml of NCTD). After treatment with NCTD for 24 h and with concentration increasing, apoptosis which presented bright blue/brown dyes even with fragment nuclei of HT-29 cells, HDLECs or the co-culture system was increased significantly as compared to control group; of them, fewer apoptosis which showed more shallow blue or brown dye in HT-29 cells or the co-culture system as compared to HDLECs. b Cell apoptosis induced by different concentration of NCTD and time-response curves via FCM. In FCM scatter diagram, the left lower quadrant shows live cells (FITC-/PI-), the right upper quadrant for necrotic or death cells (FITC+/PI+), and the right lower quadrant for apoptotic cells (FITC+/PI-)]. No apoptotic cells were observed in control group (0 μg/ml of NCTD). After treatment with NCTD for 24 h and with concentration increasing, apoptotic percent of HT-29 cells, HDLECs or the co-culture system was increased significantly in a time-dependent manner, and with decreased living HT-29 cells, HDLECs and co-culture cells, increased apoptotic cells under the right lower quadrant (FITC+/PI-), more necrotic or death cells under the right upper quadrant (FITC+/PI+) as compared to control group (P < 0.001); moreover, apoptotic percent of HT-29 cells or the co-culture system was less than that of HDLECs (*P < 0.05). c The inhibitory effect of NCTD on expression of anti-apoptotic gene Bcl-2 in HDLECs and the co-culture system in vitro. The positive expression, with brown-yellow dye, of Bcl-2 protein product occurred in cytoplasm. After treatment with 1/3 IC50 NCTD for 48 h, the positive index of Bcl-2 protein expression in HDLECs (0.556 ± 0.066 vs. 0.108 ± 0.039) or the co-culture system (0.702 ± 0.098 vs. 0.221 ± 0.048) was respectively decreased significantly as compared to control group (all P < 0.001), and the dye of cell nucleoli became light and shallow