Fig. 5

Cirsiliol-induced apoptosis is associated with autophagy in OS cells. A U2OS or U2OS/MTX cells were treated with DMSO (10 and 20 µM) at three concentrations and lysates were extracted, and western blot analysis was used to measure protein expression of LC3B, BECN1 and ACTB. B Immunofluorescence analysis of the number of LC3B spots formed by U2OS or U2OS/MTX cells after treatment with DMSO (20 µM). Scale bar: 50 µm. C Representative statistical analysis graph of B using FIJI/ImageJ. Five to 10 fields of view were taken, and three replicate experiments were performed. D Graphical representation of the variance in autophagy levels between U2OS and U2OS/MTX cells treated with an equivalent concentration of cirsiliol. E Lysosomes were labeled using LTG and then co-stain with MTRC to observe changes in the mitochondrial structure and the number of lysosomes in U2OS or U2OS/MTX cells. Scale bar: 50 µm. F, G. U2OS or U2OS/MTX cells were treated with permutations of cirsiliol and/or EBSS for 48 h. The proportion of apoptosis was detected by Annexin-V-FITC staining. Data are expressed as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001