CDKs targeted by dinaciclib and their related cyclin protein expression in NT2/D1, NT2/D1-R, NCCIT and NCCIT-R cells. A total of 30 µg of total cell lysate was separated on a 4–12% Bis–Tris gel as described. Lane 1—NT2/D1, lane 2—NT2/D1-R, lane 3—NCCIT, lane 4—NCCIT-R. The human β-tubulin was used as an internal control. Quantification results are presented as a relative optical density means ± SEM of three independent experiments, and representative Western blot results are shown. * p < 0.0001, # p < 0.001, § p < 0.05 vs. sensitive subclones.

Effect of dinaciclib on NT2/D1/-R and NCCIT/-R cells. (a) Cell viability. NT2/D1/-R (1) and NCCIT/-R (2) cells were treated with increasing concentrations of dinaciclib for forty-eight hours as described in Methods. Cell viability was analyzed using the MTT assay. Results are expressed as the percentage of viable cells vs. untreated cells (ctrl). Data are the mean ± S.E.M. of three experiments performed in triplicate. * p < 0.0001 vs. untreated cells, # p < 0.001 vs. untreated cells. (b) Cell proliferation. NT2/D1/-R (1b) and NCCIT/-R (2b) were treated for forty-eight hours with the corresponding calculated IC25, IC50 and IC80 concentrations of dinaciclib. Cell proliferation was assessed using the cytometer count. Results are expressed as the percentage of viable cells vs. untreated cells (Ctrl) ± SEM. § p < 0.05 vs. untreated cells, # p < 0.001 vs. untreated cells, * p < 0.0001 vs. untreated cells. (c) NT2/D1/-R and NCCIT/-R cell-cycle distribution after drug treatment. NT2/D1/-R (1c) and NCCIT/-R (2c) were treated with the corresponding IC50 concentration of dinaciclib for forty-eight hours, and the cell-cycle distribution was analyzed. Histograms representing the percentage of cells in each cell-cycle phase are reported. § p < 0.05 vs. untreated cells, # p < 0.001 vs. untreated cells.

Effect of CP plus dinaciclib treatment on NT2/D1/-R cells. (a) Cell viability. NT2/D1 (1a) and NT2/D1-R (2a) cells were treated with increasing concentrations of CP alone or in the presence of the corresponding IC₂₅/IC₅₀ dose of dinaciclib (DIN). After forty-eight hours of treatment, cell viability was measured using the MTT assay. Results are expressed as the percentage of viable cells vs. untreated cells (Ctrl) ± SEM. ° p < 0.05 vs. untreated cells, § p < 0.01 vs. untreated cells; # p < 0.001 vs. untreated cells, * p < 0.0001 vs. untreated cells. (b) Cell-cycle analysis. NT2/D1 (1b) and NT2/D1-R cells (2b) were treated with the corresponding IC50 concentration of CP/CP plus dinaciclib for forty-eight hours, and the cell-cycle distribution was analyzed. Histograms representing the percentage of cells in each cell-cycle phase are reported. # p < 0.001 vs. untreated cells, * p < 0.0001 vs. untreated cells.

Effect of CP plus dinaciclib treatment on NT2/D1/-R cells. (a) Cell viability. NT2/D1 (1a) and NT2/D1-R (2a) cells were treated with increasing concentrations of CP alone or in the presence of the corresponding IC₂₅/IC₅₀ dose of dinaciclib (DIN). After forty-eight hours of treatment, cell viability was measured using the MTT assay. Results are expressed as the percentage of viable cells vs. untreated cells (Ctrl) ± SEM. ° p < 0.05 vs. untreated cells, § p < 0.01 vs. untreated cells; # p < 0.001 vs. untreated cells, * p < 0.0001 vs. untreated cells. (b) Cell-cycle analysis. NT2/D1 (1b) and NT2/D1-R cells (2b) were treated with the corresponding IC50 concentration of CP/CP plus dinaciclib for forty-eight hours, and the cell-cycle distribution was analyzed. Histograms representing the percentage of cells in each cell-cycle phase are reported. # p < 0.001 vs. untreated cells, * p < 0.0001 vs. untreated cells.

Effect of CP plus dinaciclib treatment on NCCIT/-R cells. (a) Cell viability. NCCIT (1a) and NCCIT-R (2a) cells were treated with increasing concentrations of CP alone or in the presence of the corresponding IC₂₅/IC₅₀ dose of dinaciclib (DIN). After forty-eight hours of treatment, cell viability was measured using the MTT assay. Results are expressed as the percentage of viable cells vs. untreated cells (Ctrl) ± SEM. ° p < 0.05 vs. untreated cells, § p < 0.01 vs. untreated cells; # p < 0.001 vs. untreated cells, * p < 0.0001 vs. untreated cells. (b) Cell-cycle analysis. NCCIT (1b) and NCCIT-R cells (2b) were treated with the corresponding IC50 concentration of CP/CP plus dinaciclib for forty-eight hours, and the cell-cycle distribution was analyzed. Histograms representing the percentage of cells in each cell-cycle phase are reported. ° p < 0.05 vs. untreated cells, # p < 0.001 vs. untreated cells, * p < 0.0001 vs. untreated cells.

Area of NT2/D1/-R and NCCIT/-R tumor xenograft in AB zebrafish embryos exposed to dinaciclib alone or combined with CP. (a) Tumor areas at 120 hpf (T3—end of treatment) of drug-treated and vehicle-treated embryos were measured using Zen 2.3 Black software from ZEISS. Results are reported as area (µm²) ± SEM. * p < 0.0001, § p < 0.01, ° p < 0.05 vs. vehicle or single drug as indicated in Table 3. (b) Representative, lateral-view pictures of Tg (kdrl:EGFP) untreated and treated embryos at 120 hpf xenografted with NT2/D1 cells. Cells were labeled with a red fluorescent lipophilic dye, while the embryo endothelium was labeled with a green fluorescent protein reporter driven by the kdrl promoter. Images were acquired using a Zeiss LSM 900 confocal microscope at 10× magnification.