Fig. 5
Analysis of pro-apoptotic effects of AM on HeLa cells. a Representative plots obtained by flow cytometry using Annexin A5 Apoptosis Detection kit were used to quantify cell viability and apoptosis following treatment with AM at IC50 and IC90 concentrations for 12 h. b Proportion of viable cells, early and late apoptotic cells, and dead cells analyzed using flow cytometry. c Representative TUNEL assay images captured at a magnification of 20 × , where brown cells indicate positive DNA fragmentation. d Fluorescent images of DAPI staining observed at 60 × magnification indicate nuclear fragmentation. e Flow cytometry using the DCFH-DA probe indicated the distribution of cell groups treated with AM at IC50 and IC90 concentrations compared with the non-treated cells, reflecting intracellular ROS levels in HeLa cells. f The average intensity of DCFH-DA supported the observed trend of increased ROS levels with AM treatment. g Western blot images of caspase 3, caspase 9, and pro-apoptotic (BAX, BAK) and anti-apoptotic markers (BCL2, BCL-XL) in HeLa cells treated with IC50 and IC90 doses of AM for 12 h. The representative images are cropped from larger membranes. Original images of blots are shown in Fig. S9. h Relative expression levels of pro-apoptotic (BAX, BAK) and anti-apoptotic markers (BCL2, BCL-XL) of AM-treated group to that of the non-treated group, calculated from the intensities of bands obtained by western blotting. i Ratio of expression levels of pro-apoptotic to those of anti-apoptotic proteins (BAX/BCL2 and BAK/BCL-XL). IC50, 2.6 µg/mL; IC90, 6.25 µg/mL. Data are represented as mean ± SD and statistically analyzed by one-way ANOVA. Statistically significant differences were observed between the treated and the non-treated groups; *, p < 0.05; #, p < 0.01; †, p < 0.001; n = 3. Scale bars indicate 50 μm