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X. Li et al.
ly increased Mcl-1 protein levels compared to Z-VAD- FMK alone (Online Supplementary Figure S3). Using the parental compounds of CUDC-907, SAHA (a pan-HDAC inhibitor) and GDC-0941 (a PI3K inhibitor), alone or com- bined (at a 1:1 ratio) at inhibitory concentrations, we found that SAHA and GDC-0941 synergistically induced apoptosis (Online Supplementary Figure S4A, C). The half maximal effective concentrations (EC50) were much higher for the combination of SAHA plus GDC-0941 than for CUDC-907, suggesting that while they do synergize, the hybrid is more potent than the parental compounds.
A
Western blotting analysis of whole cell lysates from MOLM-13 cells treated with CUDC-907 for up to 24 h, revealed decreased Mcl-1 and increased Bim, cleaved cas- pase 3, and cleaved PARP at the 12 h time-point (Figure 4A). Decreased RRM1 and CHK1, and increased γH2AX were also detected at the 12 h time-point, while decreased Wee1 was detected as early as 4 h after CUDC-907 treat- ment (Figure 4B). Similar protein level changes were detected 16 h after treatment in U937 cells (Online Supplementary Figure S5). Annexin V/PI staining revealed a significant increase in annexin V+ cells, indicating that
B
CD
Figure 4. Upregulation of Bim and downregulation of Mcl-1, CHK1, Wee1, and RRM1 coincide with induc- tion of apoptosis. (A, B) MOLM-13 cells were treat- ed with CUDC-907 for up to 24 h. Whole cell lysates were subjected to western blotting and probed with the indicated antibodies. The fold changes for the densitometry measure- ments, normalized to β- actin and then compared to no drug control, are indi- cated below the correspon- ding blot. Cells treated with 50 nM CUDC-907 for 24 h were used as a positive control for γH2AX and cleaved caspase 3. (C, D) U937 (C) and MOLM-13 (D) cells were treated with CUDC-907 for up to 24 h and then subjected to annexin V-FITC/propidium iodide (PI) staining and flow cytometry analyses. Mean percent annexin V+ cells ± standard error of mean are shown. **P<0.01, ***P<0.001. Bim S, L, and EL indicate Bim short, long, and extra- long isoforms, respectively. CUDC: CUDC-907; cf- Caspase 3: cleaved cas- pase 3; cf-PARP: cleaved PARP.
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haematologica | 2019; 104(11)