Fig. 5.

CBX inhibits FOXO3-mediated SESN3 expression and associated chemoprotection in high-stage NB cells. a Clonogenic survival of NB4/FOXO3 and NB8/FOXO3 cells was analyzed by CFA. The cells were treated for 72 hours with 50 nM 4OHT and 80 µM CBX alone and for further 72 hours with etoposide and doxorubicin in combination. A concentration of 0.1 µg/ml etoposide or 0.01 µg/ml doxorubicin were added to NB4/FOXO3 cells and 0.8 µg/ml etoposide or 0.08 µg/ml doxorubicin to NB8/FOXO3 cells. Colonies were stained with crystal violet. b Quantification of the CFA was performed by photometric measurement after discoloration with 0.5% SDS in 50% ethanol. Shown are means ± s.e.m. of at least three independent experiments; *P < 0.05, **P < 0.025, ***P < 0.01. c NB8/FOXO3 cells were treated with 100 nM 4OHT alone or in combination with 120 µM CBX for six hours and quantitative RT-PCR of SESN3 expression was performed. Shown are mean values ± s.e.m. of three independent experiments. Statistical analyses were done with the Student’s unpaired t test; *P < 0.05, **P < 0.025 compared with controls. d Immunoblot analyses of SESN3, P27KIP1, and FOXP1 expression in NB8/FOXO3 cells treated with 50 nM 4OHT and the indicated concentrations of CBX for 24 hours. GAPDH served as loading control