Figure 7.
Effect of BIX01294 on MM cell sensitivity to PIs. (A-C) Effect of G9a/GLP targeting on carfilzomib-induced cell death. (A) Effect of carfilzomib and/or BIX01294 (BIX-1294) treatment on human cell lines. Human OPM-2 (lower panel) and XG-7 (upper panel) cells were treated with the indicated concentrations of carfilzomib and/or BIX-1294 for 4 days. The viability was determined using a CellTiter-Glo assay, and synergy scores were calculated using the Bliss method. Results are the mean of 3 independent experiments. (B) Effect of G9a knockdown on carfilzomib-induced cell death. Transduced XG-7 shG9a#1 cells were cultured with increasing doses of carfilzomib 3 days after treatment or not with doxycycline (Dox) for an additional 4 days. The viability was determined using a CellTiter-Glo assay. Results are mean ± standard deviation of 4 independent experiments. *P < .05, **P < .005, ***P < .001. (C) Effect of carfilzomib (Cfz) and/or BIX01294 (BIX) treatment on primary human CD138+ MM cells. Mononuclear cells from 7 MM patients were treated with the indicated concentrations for 4 days, and the percentage of viable CD138+ plasma cells was determined by flow cytometry. Results are expressed as the relative viability compared with control (Cnt). (D) Western blot analysis of the mechanisms underlying the increased carfilzomib (Cfz) sensitivity upon BIX01294 (BIX) treatment. XG-7 cells were treated with BIX01294 (2 µM) and/or carfilzomib (2 nM) for 24 hours, after which western blot analysis was performed on whole-cell lysates for the indicated proteins. Actin was used as loading control, and 1 experiment representative of 3 experiments is shown. (E-F) Effect of BIX01294 (BIX), bortezomib (Bz), and combination (Combo) treatment on tumor burden (E) and survival (F) in the murine 5TGM1 model. Mice were inoculated with 5TGM1 cells and assigned to different treatment groups: vehicle, 10 mg/kg (mpk) BIX, 0.6 mg/kg (mpk) Bz, or BIX plus Bz (Combo). The number of mice per treatment group and the treatment scheme are indicated in the schematic overview (upper panel). To determine the effect on tumor burden (E), all mice were euthanized when the first mouse showed obvious signs of morbidity. *P < .05, ***P < .001. (F) To determine the effect on OS, each mouse was euthanized individually when showing clear signs of morbidity, and the effect on survival rates was determined by Kaplan-Meier analysis. *P < .05 vs treatment with Bz alone.

Effect of BIX01294 on MM cell sensitivity to PIs. (A-C) Effect of G9a/GLP targeting on carfilzomib-induced cell death. (A) Effect of carfilzomib and/or BIX01294 (BIX-1294) treatment on human cell lines. Human OPM-2 (lower panel) and XG-7 (upper panel) cells were treated with the indicated concentrations of carfilzomib and/or BIX-1294 for 4 days. The viability was determined using a CellTiter-Glo assay, and synergy scores were calculated using the Bliss method. Results are the mean of 3 independent experiments. (B) Effect of G9a knockdown on carfilzomib-induced cell death. Transduced XG-7 shG9a#1 cells were cultured with increasing doses of carfilzomib 3 days after treatment or not with doxycycline (Dox) for an additional 4 days. The viability was determined using a CellTiter-Glo assay. Results are mean ± standard deviation of 4 independent experiments. *P < .05, **P < .005, ***P < .001. (C) Effect of carfilzomib (Cfz) and/or BIX01294 (BIX) treatment on primary human CD138+ MM cells. Mononuclear cells from 7 MM patients were treated with the indicated concentrations for 4 days, and the percentage of viable CD138+ plasma cells was determined by flow cytometry. Results are expressed as the relative viability compared with control (Cnt). (D) Western blot analysis of the mechanisms underlying the increased carfilzomib (Cfz) sensitivity upon BIX01294 (BIX) treatment. XG-7 cells were treated with BIX01294 (2 µM) and/or carfilzomib (2 nM) for 24 hours, after which western blot analysis was performed on whole-cell lysates for the indicated proteins. Actin was used as loading control, and 1 experiment representative of 3 experiments is shown. (E-F) Effect of BIX01294 (BIX), bortezomib (Bz), and combination (Combo) treatment on tumor burden (E) and survival (F) in the murine 5TGM1 model. Mice were inoculated with 5TGM1 cells and assigned to different treatment groups: vehicle, 10 mg/kg (mpk) BIX, 0.6 mg/kg (mpk) Bz, or BIX plus Bz (Combo). The number of mice per treatment group and the treatment scheme are indicated in the schematic overview (upper panel). To determine the effect on tumor burden (E), all mice were euthanized when the first mouse showed obvious signs of morbidity. *P < .05, ***P < .001. (F) To determine the effect on OS, each mouse was euthanized individually when showing clear signs of morbidity, and the effect on survival rates was determined by Kaplan-Meier analysis. *P < .05 vs treatment with Bz alone.

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