Fig. 4

NHPI induces apoptosis via mitochondrial pathway. a BT-20 cells were treated with NHPI at 2.5, 5 and 10 μM for 48 h. LoVo cells were incubated with NHPI at 5, 10 and 20 μM for 48 h or 72 h. Cell apoptosis was analyzed by flow cytometry using the Annexin V-FITC and PI double staining. Representative images were presented. b Quantification of flow cytometry analysis of apoptosis. Results were presented as mean ± SD (n = 3). *p < 0.05, **p < 0.01 and ***p <0.001, difference versus 0 μM control group. c NHPI induced MMP loss in BT-20 cells. BT-20 cells were treated with NHPI at 2.5, 5 and 10 μM for 48 h, stained with JC-1 and subjected to flow cytometry analysis. The dot-plot representation of the flow cytometry analysis shows the distribution of JC-1 aggregates (cells emitting red fluorescence detected in the FL2 channel) and JC-1 monomers (cells emitting green fluorescence detected in the FL1 channel). d Histograms showing the percentage of JC-1 aggregate-positive and JC-1 monomer-positive cells. Results were presented as mean ± SD (n = 3). **p < 0.01 and ***p <0.001, difference versus 0 μM control group. e Effect of NHPI on the expressions of apoptosis-related proteins. Cells were treated with indicated concentrations of NHPI for 24 h, followed by western blot analysis with indicated antibodies. β-actin was used as a loading control