Fig. 4

Knockdown of HSP90AA1 inhibits autophagy during chemotherapy in osteosarcoma cells. a MG-63 osteosarcoma cells were transfected with control shRNA/HSP90AA1 shRNA for 48 h and then were treated with Cis (20 μmol/L) or Dox (0.2 μg/mL) for 24 h. The protein levels of LC3 and p62 were assessed by Western blot. b Control shRNA/HSP90AA1 shRNA transfected MG-63 cells were treated with Dox (0.2 μg/mL) for 24 h with or without bafilomycin A1 (100 nmol/L). LC3 and p62 expression were analyzed by Western blot. c and d control shRNA/HSP90AA1 shRNA transfected MG-63 cells were treated with Cis (20 μmol/L) or Dox (0.2 μg/mL) for 24 h. Then autophagic flux were analyzed by mRFP-GFP-LC3 construct (c) and transmission electron microscopy (d), autophagosome-like structures were indicated (red arrows). e and f Control shRNA/HSP90AA1 shRNA transfected MG-63 cells were pretreated with rapamycin (100 nmol/L) for 6 h and then were exposed to Dox (0.2 μg/mL) for an additional 24 h. Apoptosis was analyzed by measuring Annexin V-PE/PI positive cells by flow cytometric (e, n = 3; *, p < 0.05, versus rapamycin untreated group). Autophagy was determined by measuring LC3 puncta formation (f, n = 3; *, p < 0.05 versus rapamycin untreated group). NS, not significant