Fig. 4

BTFPTU causes osteosarcoma cells death through multiple pathways in vitro. (A) Immunofluorescence of intracellular chloride ions in HOS cells treated with or without BTFPTU (2 µM) treatment for 6 h using MQAE staining. Scale bar, 200 μm. (B) The expression of ER stress proteins was measured by Western blot analysis in HOS and 143B cells treated with varying concentrations of BTFPTU for 24 h. (C) The expression of autophagy-related proteins was measured by Western blot analysis in HOS and 143B cells treated with varying concentrations of BTFPTU for 24 h. (D) The expression of apoptosis proteins was measured by Western blot analysis in HOS and 143B cells treated with varying concentrations of BTFPTU for 24 h. (E) Flow cytometry to detect cell cycle of HOS and 143B cells with or without BTFPTU (2 µM) treatment for 12 h by PI staining. (F) Quantification of different cell cycle phases of HOS and 143B cells. (G) Cell viability of HOS and 143B cells with Z-VAD (10 µM), BI-6 C (5 µM), AC (5 µM), MCC (10 µM), Fer-1 (5 µM), Lip-1 (2.5 µM), CQ (2.5 µM), 3-MA (2.5 mM) or nec-1 (5 µM) treatment under PTU, TFPTU or BTFPTU treatment for 24 h using Cell Counting Kit-8 assay. The data represent the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 for a comparison with the control group or as indicated