Fig. 3

Apatinib induced ER stress and autophagy in ESCC cells. a Electron microscopy was used to view changes in the rough endoplasmic reticulum, formation of vacuolization and the number of autophagosomes and/or autolysosomes. N nucleus, M mitochondria, RER rough endoplasmic reticulum, ASS autolysosome, GO Golgi apparatus, LD lipid droplet. b–d Western blot analysis was performed to measure the expression levels of ER stress markers after cells were incubated with apatinib for 24 h. e–g The level of cytosolic calcium was evaluated by flow cytometry. h–j The expression levels of autophagy markers in cells incubated with apatinib for 24 h were measured by western blotting. k–m The expression levels of LC-3, P62, and GAPDH in cells treated with 25 μM apatinib for 24 h or 50 nM BafA1 for 12 h were measured by western blotting. n–r Representative images and quantification of autophagosomes shown as yellow dots and autolysosomes shown as red dots were shown in cells transfected with RFP-GFP-LC3 lentivirus and incubated with 25 μM apatinib for 24 h. * p < 0.05, ** p < 0.01, *** p < 0.001 compared with control group