Figures & data
Figure 1. Continuous carfilzomib and TG02 co-treatment results in at least additive cell death. Cells were plated at a density of 0.25 × 106 cells/mL and treated with the indicated concentrations of (A) carfilzomib, (B) TG02 or the (C) combination for 24 hours. (D) Cells were co-treated with carfilzomib (IC10 and IC50 concentrations) and TG02 for 1 hour, washed and then treated with TG02 for 23 hours. Cell death was determined via Annexin–V–FITC/PI flow cytometry. Data are presented as mean +/− SEM of at least 3 independent experiments. IC50 and IC10 significance are indicated above and below the mean values, respectively. (E) Ficoll isolated buffy coat from a myeloma patient BM aspirate was collected and washed with PBS. Plasma cells were either treated in the presence of buffy coat cells (MM53 and MM55) or CD138+ plasma cells were isolated and treated (MM54). Twenty-four hour apoptosis was determined by staining with anti-CD38, anti-CD45, and Annexin V-FITC. *p > 0.05, **p > 0.01, ***p > 0.001.
Figure 2. Co-culture with Hs-5 cells protected against combination treatment in 3 cell lines, while addition of Hs-5 conditioned medium was protective in only RPMI-8226. Hs-5 cells were plated at a density of 0.05 × 106 cells/mL and allowed to grow for 48 h. MM cells were added to the Hs-5 cells after 48 h, 30 min prior to drug treatment. Conditioned medium was collected and filtered then diluted to 50% and added to MM cells. 24 h cell death was assessed via Annexin-V-FITC/PI (control and conditioned medium treated cells) or Annexin-V-FITC/anti-CD38-PE (Hs-5 co-cultured cells) flow cytometry. Data in are presented as mean +/− SEM of at least 3 independent experiments.
Figure 3. Treatment with carfilzomib causes an increase in NOXA mRNA and TG02 causes a decrease in Mcl-1 protein. Cells were plated at a density of 0.25 × 106 cells/mL and treated with carfilzomib, TG02 or the combination for 6 hours. RT-qPCR results for (A) pro-apoptotic and (B) anti-apoptotic mRNA expression levels compared to control were determined and presented as mean +/− SEM of at least 3 independent experiments. * p > 0.05, ** p > 0.01, *** p > 0.001. (C) Protein lysates were obtained after 6 hour carfilzomib, TG02 or the combination treatments (IC50) using RIPA buffer plus protease inhibitor cocktail. . 20 μg of protein were subjected to Western blot analysis with the antibodies shown. Numbers shown represent the quantified relative expression of Mcl-1 normalized to GAPDH.
Figure 4. The decreased in Mcl-1 protein expression is not due to CDK9 inhibition. MM.1s cells were treated with siCDK9 or si (control) for 24 hours and then treated with carfilzomib, TG02 or the combination for an additional 24 hours. (A) Knockdown of siCDK9 was determined using protein gel blot and densitometry analysis. Relative expression, normalized to actin, of the indicated proteins are shown below the corresponding bands. (B) Cell death was determined via Annexin-V-FITC/PtdIns flow cytometry. Data are presented as mean +/− SEM of at least 3 independent experiments.
Figure 5. Overexpression of Mcl-1 in RPMI-8226 cells confers significantly less protection than over expression of Bcl-2 or Bcl-xL.RPMI-8226 cells overexpressing the anti-apoptotic proteins shown were treated with increasing doses of (A) carfilzomib (B) TG02 or (C) the combination for 24 hours. Cell death was determined via Annexin-V-FITC/PI flow cytometry. Data are presented as mean +/− SEM of at least 3 independent experiments. Statistical analysis for RPMI-8226 Mcl-1 over expressing cells only shown. *p > 0.05, **p > 0.01, ***p > 0.001.
