Figure 2.
I-BET762 shows a synergistic effect with copanlisib by suppressing ATL proliferation and downregulating the expression of c-MYC. (A) The combined effects of I-BET762 with copanlisib inhibitors on ATL43Tβ (-) cell line incubated for 72 hours in the presence of 5 increasing concentrations of I-BET762 (31.2 to 500 nM, blue), copanlisib (7.8 to 125 nM, orange), or their combinations (green). A thymidine incorporation assay was performed, and the percentages of control were calculated from the counts per minute (CPM) of samples with drugs divided by the CPM of samples with no inhibitor × 100 (black bar). A representative figure of 3 independent experiments is shown. (B) CI plot of I-BET762 and copanlisib combination in ATL43Tβ (-) cell line. CI dots represent 5 combination data points, CI <1 was considered synergism. (C) Immunoblot analysis of c-MYC, p-AKTS473, p-4EBP1 after 1 hour of incubation with either 1 μM of I-BET762 or copanlisib or in combination. For p27 expression, cells were incubated for 24 hours prior to western blot analysis. (D) Induction of cell apoptosis by I-BET762 and copanlisib combination. ATL43Tβ (-), ST1, ED41214 (-), and Jurkat cells were treated with either DMSO, 1 μM of I-BET762, or copanlisib or in combination for 48 hours, and the cell apoptosis was measured with annexin V staining and analyzed by flow cytometry. (E) The bar graphs represent the percentages of early apoptotic cells (annexin V+7AAD−, lower right quadrant) and (F) late apoptosis (annexin V+7AAD+, upper right quadrant), n = 3. (G,H) Flow cytometric analysis of active caspase3 and cleaved-PARP in ATL43Tβ (-), ST1, and MT1 cell lines. The cells were treated with inhibitors for 48 hours and stained for the double-positive cells of the percentage of caspase 3+cleaved-PARP+ represented in the bar graphs (n = 3). Human fibroblast cells were used as a control. Three biological experiments were performed, and values are presented as mean ± SEM. One-way ANOVA was used to determine statistical differences. *P < .05, **P < .01, ***P < .001, ****P < .0001.

I-BET762 shows a synergistic effect with copanlisib by suppressing ATL proliferation and downregulating the expression of c-MYC. (A) The combined effects of I-BET762 with copanlisib inhibitors on ATL43Tβ (-) cell line incubated for 72 hours in the presence of 5 increasing concentrations of I-BET762 (31.2 to 500 nM, blue), copanlisib (7.8 to 125 nM, orange), or their combinations (green). A thymidine incorporation assay was performed, and the percentages of control were calculated from the counts per minute (CPM) of samples with drugs divided by the CPM of samples with no inhibitor × 100 (black bar). A representative figure of 3 independent experiments is shown. (B) CI plot of I-BET762 and copanlisib combination in ATL43Tβ (-) cell line. CI dots represent 5 combination data points, CI <1 was considered synergism. (C) Immunoblot analysis of c-MYC, p-AKTS473, p-4EBP1 after 1 hour of incubation with either 1 μM of I-BET762 or copanlisib or in combination. For p27 expression, cells were incubated for 24 hours prior to western blot analysis. (D) Induction of cell apoptosis by I-BET762 and copanlisib combination. ATL43Tβ (-), ST1, ED41214 (-), and Jurkat cells were treated with either DMSO, 1 μM of I-BET762, or copanlisib or in combination for 48 hours, and the cell apoptosis was measured with annexin V staining and analyzed by flow cytometry. (E) The bar graphs represent the percentages of early apoptotic cells (annexin V+7AAD−, lower right quadrant) and (F) late apoptosis (annexin V+7AAD+, upper right quadrant), n = 3. (G,H) Flow cytometric analysis of active caspase3 and cleaved-PARP in ATL43Tβ (-), ST1, and MT1 cell lines. The cells were treated with inhibitors for 48 hours and stained for the double-positive cells of the percentage of caspase 3+cleaved-PARP+ represented in the bar graphs (n = 3). Human fibroblast cells were used as a control. Three biological experiments were performed, and values are presented as mean ± SEM. One-way ANOVA was used to determine statistical differences. *P < .05, **P < .01, ***P < .001, ****P < .0001.

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