Fig. 4

AMG9810 enhances the response to bortezomib in MM cells. a, b MM cells were exposed to low-dose bortezomib (BTZ) (3 nM) in the absence or presence of AMG9810 (10 µM) for 48 h. a Apoptosis was assessed using Annexin V/PI staining and flow cytometry analysis. Percentage of early (Annexin V+/PI−) and late (Annexin V+/PI+) apoptotic cells is presented as mean of triplicates ± SD (**p < 0.01). b Cell cycle distribution was determined using 7-AAD staining. Percent of proliferating cells (G2/M + S fractions), non-proliferating cells (G0/G1 fraction) and cells with fragmented DNA (sub G0/G1 fraction) are presented as mean of triplicates ± SD (**p < 0.01). c Effect of treatment with bortezomib (4 nM for RPMI8226-EV, 6 nM for RPMI8226-CXCR4), AMG9810 (10 µM) or their combination on the viability of MM cells incubated in the absence or presence of BMSCs, measured by PI-exclusion and flow cytometry analysis. d Effect of AMG9810 on BMSC-induced intra-cellular signaling in RPMI8226-CXCR4. RPMI8226-CXCR4 cells were treated with AMG9810 (10 µM), bortezomib (10 nM) or their combination in the absence or presence of BMSC for 24 h. Levels of phosphorylated AKT and pS6 proteins, measured by Western blot, β-actin were used as internal control. e RPMI8226-CXCR4 and RPMI8226-CXCR4-bortezomib-resistant cells were incubated during 48 h with indicated concentrations of bortezomib or AMG9810. Viability was evaluated using the XTT method. Results represent the average of triplicates ± SD (**p < 0.01). f RPMI8226-CXCR4 and RPMI8226-CXCR4-Bort^Res cells were exposed to bortezomib (BTZ) (10 nM) and carfilzomib (CFZ) (25 nM) in the absence or presence of AMG9810 (10 µM) for 48 h. Apoptosis was assessed using Annexin V/PI staining and flow cytometry analysis. Percentage of early (Annexin V+ /PI−) and late (Annexin V+/PI+) apoptotic cells is presented as mean of triplicates ± SD (**p < 0.01)