Figure 3.
MDM2 inhibition increases p53 binding to TRAIL-R1/2 promoters and sensitizes AML cells to TRAIL-mediated apoptosis. (A) The graph shows the percentage of viable cells. Where indicated, WT OCI-AML3 or p53 KD OCI-AML3 were incubated with 1 µM MDM2 inhibitor RG-7112. After 48 hours, limiting concentrations of hTRAIL (TNFSF 10) were added for 24 hours, where indicated. The viability of cells was measured by flow cytometry. The mean of triplicates ± standard error of the mean (SEM) is displayed. P values were calculated using a 2-sided Student unpaired t test. (B-C) ChIP-qPCR analysis in OCI-AML3 cells treated with DMSO or 2 µM RG-7112 for 12 hours to detect the binding of p53 to the promoter of (B) TRAIL-R1 (TNFRSF10A) and (C) TRAIL-R2 (TNFRSF10B). Data are represented as percent input and are representative of 3 experiments; error bars, SEM from 3 technical replicates. N.D., not detected. (D) The bar diagram shows the viability of WT or TRAIL-R2−/− OCI-AML3 cells (TRAIL-R2−/−) that were incubated with 1 µM of the MDM2 inhibitor RG-7112, where indicated. After 48 hours, limiting concentrations of hTRAIL (TNFSF 10) were added for 24 hours, where indicated. The viability of the AML cells was measured by flow cytometry. The mean of triplicates ± SEM is displayed. P values were calculated using a 2-sided Student unpaired t test. (E) Representative Western blots showing caspase-8, caspase-3, PARP, and loading control (β-actin) in human OCI-AML3 cells. OCI-AML3 cells exposed to DMSO or RG-7112 (1 µM) were cocultured with activated T cells at an E:T ratio of 10:1 for 4 hours. (F) The bar diagram shows the viability of MOLM-13 cells pretreated with DMSO or 1 µM MDM2 inhibitor for 24 hours, as indicated. hTRAIL (TNFSF10) was added for an additional 24 hours before analysis by flow cytometry. P values were calculated using the one-way ANOVA test. (G) The graph shows the viability of MV4-11 cells pretreated with 1 µM MDM2 inhibitor for 24 hours. Limiting concentrations of hTRAIL (TNFSF10) were added for an additional 24 hours before analysis by flow cytometry. P values were calculated using the one-way ANOVA test. ns, not significant.

MDM2 inhibition increases p53 binding to TRAIL-R1/2 promoters and sensitizes AML cells to TRAIL-mediated apoptosis. (A) The graph shows the percentage of viable cells. Where indicated, WT OCI-AML3 or p53 KD OCI-AML3 were incubated with 1 µM MDM2 inhibitor RG-7112. After 48 hours, limiting concentrations of hTRAIL (TNFSF 10) were added for 24 hours, where indicated. The viability of cells was measured by flow cytometry. The mean of triplicates ± standard error of the mean (SEM) is displayed. P values were calculated using a 2-sided Student unpaired t test. (B-C) ChIP-qPCR analysis in OCI-AML3 cells treated with DMSO or 2 µM RG-7112 for 12 hours to detect the binding of p53 to the promoter of (B) TRAIL-R1 (TNFRSF10A) and (C) TRAIL-R2 (TNFRSF10B). Data are represented as percent input and are representative of 3 experiments; error bars, SEM from 3 technical replicates. N.D., not detected. (D) The bar diagram shows the viability of WT or TRAIL-R2−/− OCI-AML3 cells (TRAIL-R2−/−) that were incubated with 1 µM of the MDM2 inhibitor RG-7112, where indicated. After 48 hours, limiting concentrations of hTRAIL (TNFSF 10) were added for 24 hours, where indicated. The viability of the AML cells was measured by flow cytometry. The mean of triplicates ± SEM is displayed. P values were calculated using a 2-sided Student unpaired t test. (E) Representative Western blots showing caspase-8, caspase-3, PARP, and loading control (β-actin) in human OCI-AML3 cells. OCI-AML3 cells exposed to DMSO or RG-7112 (1 µM) were cocultured with activated T cells at an E:T ratio of 10:1 for 4 hours. (F) The bar diagram shows the viability of MOLM-13 cells pretreated with DMSO or 1 µM MDM2 inhibitor for 24 hours, as indicated. hTRAIL (TNFSF10) was added for an additional 24 hours before analysis by flow cytometry. P values were calculated using the one-way ANOVA test. (G) The graph shows the viability of MV4-11 cells pretreated with 1 µM MDM2 inhibitor for 24 hours. Limiting concentrations of hTRAIL (TNFSF10) were added for an additional 24 hours before analysis by flow cytometry. P values were calculated using the one-way ANOVA test. ns, not significant.

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