Fig. 6

The role of autophagy induction in the response to each treatment in OS cells. HOS cells were pretreated with Baf-A1 (100 nM) for 2 h prior to treatment with CAP (100 μM) and DDP (16.7 μM) alone or in combination for 24 h. The expression levels of autophagy-related and apoptosis-related proteins were measured by Western blotting (a). Statistical analyses of LC3II/LC3I (b), p62 (c), Bcl-2 (d), Bax (e), and cleaved caspase-3 (f) in HOS cells. The relative cell viability (%) was determined using a CCK-8 assay (g). Apoptosis in HOS cells was determined by flow cytometry after staining with Annexin V-FITC/PI (h, i). The quantitative data are shown as the mean ± SD of 3 independent experiments; *p < 0.05, **p < 0.01 and ***p < 0.001 vs. the control (CAP-, DDP-, Baf-A1-). The role of reactive oxygen species (ROS) generation in HOS cells after CAP/DDP combination treatment was assessed. HOS cells were pretreated with NAC (5 mM) for 2 h prior to cotreatment with CAP (100 μM) and DDP (16.7 μM) for another 24 h. The levels of cellular ROS were determined by fluorescence microscopy and flow cytometry after DCFH-DA (10 mM) staining (j, k). The relative cell viabilities (%) were determined using the CCK-8 assay (l). The apoptosis percentages were determined by flow cytometry after staining with Annexin V-FITC/PI (M, N). After ROS scavenging by NAC, the expression levels of proteins involved in autophagy induction, apoptosis, the ROS/JNK signaling pathway and the AKT/mTOR signaling pathway were measured by Western blotting (o). Statistical analyses of p-AKT (p), p-mTOR (q), p-JNK (r), beclin1 (s), LC3II/LC3I (t), Bcl-2 (u), Bax (v), and cleaved caspase-3 (w) in HOS cells. The quantitative data are shown as the mean ± SD of 3 independent experiments; *p < 0.05, **p < 0.01 and ***p < 0.001 vs. the control (CAP+DDP-, NAC-)