Figure 4.
In vitro functional sequelae observed in MM cells exposed to AZD4785. (A) MM.1S, MM.1R, RPMI.8226, OPM2, KMS12, and U266 cells were exposed to AZD4785 or ASO control (ctrl) for 72 hours at the indicated concentrations. Cell proliferation was evaluated by CellTiter Glo. Average of triplicate experiments ± standard deviation (SD) is shown. (B) MM.1S cells were cultured in the presence or absence of AZD4785 (0-10 μM; 48 hours). MM.1R, KMS20, OPM2, and KMS12 cells were cultured in the presence or absence of AZD4785 or ASO ctrl at the indicated concentrations for 48 hours. MM cells were then harvested, and cell lysates were subjected to western blot using anti–p-ERK, –p-MEK, –p-RSK90, –p-AKT, and –glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies. (C) MM.1S cells were exposed to AZD4785 (3 μM) for 48 hours and subjected to wide transcriptome profiling, showing a significant inhibition of different cell cycle–related gene sets, as assessed by gene set enrichment analysis (GSEA). Normalized enrichment score (NES) was generated by comparing AZD4785-treated vs untreated cells. NES, nominal P value, and false discovery rate (FDR) q value are reported for each plot. (D) Cytofluorimetric analysis of cell cycle performed using AZD4785-treated MM.1S cells (48 hours). Average of triplicate experiments ± SD is shown. (E) Annexin V/proteasome inhibitor staining was performed using AZD4785-treated MM.1S cells (48 hours). Average of triplicate experiments ± SD is shown. MM.1S cells were cultured in the presence or absence of AZD4785 at the indicated concentrations for 48 hours. MM.1R, KMS20, OPM2, and KMS12 cells were cultured in the presence or absence of AZD4785 or ASO ctrl at the indicated concentrations for 48 hours. MM cells were then harvested, and cell lysates were subjected to western blot using anti-BIM, -PARP, -GAPDH, and –β-actin antibodies. (F) Cytofluorimetric analysis of mtROS production in MM1S cells exposed to AZD4785 (3 μM; 48 hours). MM.1S cells were cultured in the presence or absence of AZD4785 (0-10 μM; 48 hours). MM.1R, KMS20, OPM2, and KMS12 cells were cultured in the presence or absence of AZD4785 or ASO ctrl (0-10 μM; 48 hours). MM cells were then harvested, and cell lysates were subjected to western blot using anti-NRF2, –p-H2AX, and -GAPDH antibodies. (G) MM.1S cells were exposed to AZD4785 (0-3 μM) for 48 hours and subjected to wide transcriptome profiling, showing a significant inhibition of c-Myc– and ρ-related gene sets, as assessed by GSEA. NES was generated by comparing AZD4785-treated vs untreated cells. NES, nominal P value, and FDR q value are reported for each plot. MM.1S cells were cultured in the presence or absence of AZD4785 (0-10 μM; 48 hours). MM cells were then harvested, and cell lysates were subjected to western blot using anti–c-Myc, -RhoA, and –α-tubulin antibodies. n.s., not significant.

In vitro functional sequelae observed in MM cells exposed to AZD4785. (A) MM.1S, MM.1R, RPMI.8226, OPM2, KMS12, and U266 cells were exposed to AZD4785 or ASO control (ctrl) for 72 hours at the indicated concentrations. Cell proliferation was evaluated by CellTiter Glo. Average of triplicate experiments ± standard deviation (SD) is shown. (B) MM.1S cells were cultured in the presence or absence of AZD4785 (0-10 μM; 48 hours). MM.1R, KMS20, OPM2, and KMS12 cells were cultured in the presence or absence of AZD4785 or ASO ctrl at the indicated concentrations for 48 hours. MM cells were then harvested, and cell lysates were subjected to western blot using anti–p-ERK, –p-MEK, –p-RSK90, –p-AKT, and –glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies. (C) MM.1S cells were exposed to AZD4785 (3 μM) for 48 hours and subjected to wide transcriptome profiling, showing a significant inhibition of different cell cycle–related gene sets, as assessed by gene set enrichment analysis (GSEA). Normalized enrichment score (NES) was generated by comparing AZD4785-treated vs untreated cells. NES, nominal P value, and false discovery rate (FDR) q value are reported for each plot. (D) Cytofluorimetric analysis of cell cycle performed using AZD4785-treated MM.1S cells (48 hours). Average of triplicate experiments ± SD is shown. (E) Annexin V/proteasome inhibitor staining was performed using AZD4785-treated MM.1S cells (48 hours). Average of triplicate experiments ± SD is shown. MM.1S cells were cultured in the presence or absence of AZD4785 at the indicated concentrations for 48 hours. MM.1R, KMS20, OPM2, and KMS12 cells were cultured in the presence or absence of AZD4785 or ASO ctrl at the indicated concentrations for 48 hours. MM cells were then harvested, and cell lysates were subjected to western blot using anti-BIM, -PARP, -GAPDH, and –β-actin antibodies. (F) Cytofluorimetric analysis of mtROS production in MM1S cells exposed to AZD4785 (3 μM; 48 hours). MM.1S cells were cultured in the presence or absence of AZD4785 (0-10 μM; 48 hours). MM.1R, KMS20, OPM2, and KMS12 cells were cultured in the presence or absence of AZD4785 or ASO ctrl (0-10 μM; 48 hours). MM cells were then harvested, and cell lysates were subjected to western blot using anti-NRF2, –p-H2AX, and -GAPDH antibodies. (G) MM.1S cells were exposed to AZD4785 (0-3 μM) for 48 hours and subjected to wide transcriptome profiling, showing a significant inhibition of c-Myc– and ρ-related gene sets, as assessed by GSEA. NES was generated by comparing AZD4785-treated vs untreated cells. NES, nominal P value, and FDR q value are reported for each plot. MM.1S cells were cultured in the presence or absence of AZD4785 (0-10 μM; 48 hours). MM cells were then harvested, and cell lysates were subjected to western blot using anti–c-Myc, -RhoA, and –α-tubulin antibodies. n.s., not significant.

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