Fig. 6.

Inhibition of FOXO3 by CBX sensitizes high-stage NB cells to chemotherapy. a The high-stage NB cell lines NB1 and NB8 were treated with the indicated concentrations of etoposide and doxorubicin in combination with 120 µM CBX for 72 hours. PI-FACS analyses were performed to detect apoptotic cells. Shown are mean values ± s.e.m. of three independent experiments. Statistical analysis was done with the Student’s unpaired t test; *P < 0.05, **P < 0.025, ***P < 0.01 compared with corresponding controls. b The caspase 3/7 activity assay was performed in NB1 and NB8 cells treated with 0.1 µg/ml etoposide or 0.01 µg/ml doxorubicin alone or in combination with 80 µM CBX for 48 hours. Shown are the means ± s.e.m. of three independent experiments, statistical analysis was done with the Student’s unpaired t test, ***P < 0.01 compared with corresponding controls. c Immunoblot analyses of FOXO3 expression in NB8/shCtr and NB8/shFOXO3 cells. GAPDH served as loading control. PI-FACS analyses were performed to detect apoptotic cells in NB8/shCtr and NB8/shFOXO3 cells treated with the indicated concentrations of etoposide for 72 hours. Shown are mean values ± s.e.m. of three independent experiments. Statistical analysis was done with the Student’s unpaired t test; ***P < 0.01 between drug-treated cell lines. d Immunoblot analyses of SESN3 expression in NB8 cells treated with 10 µg/ml etoposide alone or in combination with 120 µM CBX for four hours (left panel) and in NB8/shCtr and NB8/shFOXO3 cells treated with 10 µg/ml etoposide for four hours (right panel). GAPDH served as loading control. Densitometric analysis of SESN3 expression relative to GAPDH was done with the ImageJ 1.48 software. Untreated cells were set as 100%. e Representative images of NB8 spheroids formed by the hanging-drop technique for 96 hours. The cells were treated with 0.1 µg/ml etoposide in combination with 120 µM CBX for another 96 hours. Viable cells were stained with 2 µM calcein-AM for two hours at 37 °C. Quantification of viable cells in NB8 spheroids was done by the CellTiter-Glo 3D® cell viability assay. The cells were treated with indicated concentrations of etoposide in combination with 120 µM CBX for 96 hours. Shown are mean values ± s.e.m. of three independent experiments. Statistical analysis was done with the Student’s unpaired t test; *P < 0.05 between ±CBX treatment. f Representative images of NB8/shCtr and NB8/shFOXO3 spheroids formed by the hanging-drop technique for 96 hours treated for further 96 hours with 0.1 µg/ml etoposide. Viable cells were stained with 2 µM calcein-AM for two hours at 37 °C. Cell viability of NB8/shCtr and NB8/shFOXO3 cells was analyzed by the CellTiter-Glo 3D® cell viability assay. The spheroids were treated with 0.1 µg/ml etoposide for 96 hours. Shown are mean values ± s.e.m. of three independent experiments. Statistical analysis was done with the Student’s unpaired t test; **P < 0.025 between drug-treated cell lines