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. Author manuscript; available in PMC: 2019 Nov 17.
Published in final edited form as: Biochem Pharmacol. 2017 Dec 5;148:13–26. doi: 10.1016/j.bcp.2017.11.022

Fig. 1.

Fig. 1.

VS-5584 induces proliferation inhibition and caspase-independent cell death in AML cells. (A–C) AML cell lines and primary AML patient samples were treated with variable concentrations of VS-5584 for 72 h and viable cells were determined using MTT reagent. For AML cell lines, data are graphed as mean ± SEM from three independent experiments (panel A). For the patient samples, the IC50 values are means of duplicates from one experiment due to limited sample (panel B). Differences in VS-5584 IC50s between FLT3-ITD vs. Non-FLT3 ITD was calculated using the Mann-Whitney U test (p = .601; panel C). The horizontal lines indicate the median. (D, E) AML cell lines were treated with VS-5584 for 48 h and then subjected to Annexin V-FITC/PI staining and flow cytometry analysis. Representative dot plots are shown in panel D. Mean percent Annexin V+ cells ± SEM are shown in panel E. (F, G) MOLM-13 cells were treated with VS-5584 (or 100 nM CUDC-907 as a positive control) for 48 h. Western blots using whole cell lysates are shown (panel F). 5 × 105 cells were subjected to the JC-1 assay (panel G). (H) Annexin V-FITC/PI staining and flow cytometry analysis for MOLM-13 cells were treated for 48 h with 2 μM VS-5584 with or without 50 μM Z-VAD-FMK are shown. *** indicates p < .001. (I) Annexin V-FITC/PI staining and flow cytometry analysis results for MOLM-13 cells treated with 2 μM VS-5584 for 3, 6, 12, and 24 h are shown. *** indicates p < .001. (J, K) MOLM-13 cells were treated with 2 μM VS-5584 (24 h) or 0.125 mM ABT-199 (48 h) with or without 50 μM Z-VAD-FMK and then subjected to Annexin V-FITC/PI staining and flow cytometry analysis. ***indicates p < .001.