Fig. 6

Synergistic effects of OTX-015 (OTX) and TEM on NB cell growth. (a) Cell viability (MTT) assay showing dose-dependent growth effects of OTX/TEM in two MYCN-amplified NB cell lines at 72 h. Viable cells (%) is relative to DMSO-treated cells. Values represent mean ± SEM. Bar graphs show combination index (CI) analyses for the synergism of OTX and TEM in NB cell lines. (b) Analysis of cell cycle distribution in SK-N-BE2 cells treated with OTX (1 μM) and TEM (2 μM) alone or combined for 24 h. (c) Quantification of Annexin-V/PI positive apoptotic cells in two MYCN-amplified (SK-N-BE2, SK-N-DZ) NB cell lines treated with OTX (1 μM) and TEM (2 μM) alone or combined for 72 h. Values, mean ± SEM. **p < 0.01; ***p < 0.001 (Student-t-test). (d) Western blot images for the expression of key components of MYCN/mTOR signaling in two MYCN-amplified NB cell lines following treatment with OTX (1 μM) and TEM (2 μM) alone and combined for 24 h. Cyclophilin was used as the loading control in these analyses. The original uncropped images of these blots are provided in the Additional File 1 (Fig. S4). (e) Global protein synthesis measurement by OPP-incorporation following treatment with OTX (1 μM) and TEM (2 μM) alone or in combination for 24 h. CHX (50 μg/ml, 1 h) was used as a positive control for protein synthesis inhibition. Values represent mean ± SEM. **p < 0.01