Fig. 5.

Inhibition of autophagy inhibits the avermectin B1-mediated reduction in cell survival and apoptosis induction in osteosarcoma in vitro. (A) Western blot experiments were conducted to measure the expression changes in AMPK/ULK1 signaling pathway proteins after avermectin B1 treatment. (B) MNNG, MG63 and U2OS cells were treated with IC50 of avermectin B1 and 2 mM dorsomorphin alone or in combination as indicated and subjected to CCK8 assay. (C) Flow cytometry was conducted to measure the apoptotic rate of three osteosarcoma cell lines treated with IC50 of avermectin B1 and 2 mM dorsomorphin alone or in combination. (D) Quantification of the apoptosis proportion. (E) MNNG, MG63 and U2OS cells were treated with the IC50 of avermectin B1 and 2 mM dorsomorphin alone or in combination. Then, Western blotting was performed to detect the expression levels of the apoptosis-related protein caspase-9 and the cell cycle protein CDK6 in avermectin B1-treated cells. β-Actin was used as a loading control. (F) The LC3 puncta were analyzed by mRFP-GFP-LC3 construct when MNNG, MG63 and U2OS cells were treated with avermectin B1 and dorsomorphin alone or in combination. (G) Quantification of the autophagosomes (yellow dots) and autolysosomes (free red dots). (H) The protein expression levels of the AMPK/ULK1 pathway proteins BECN1 and LC3B after treatment with avermectin B1, dorsomorphin or avermectin B1 + dorsomorphin. Data are shown as the means ± SDs with scatter plots from at least three independent experiments. Statistical analysis was performed using Student’s t test. *p < 0.05, **p < 0.01, *** p < 0.001