IDR E804-induced apoptosis and sensitized MV4-11-R to ABT-869. (A) Two million cells of MV4-11-R were treated with either DMSO control or IDR E804 at concentrations of 100 and 200 nM for 48 hours. Cells were then washed and stained with annexin V–FITC for apoptosis assay. The shown graphs represent 3 independent experiments. (B) MV4-11-R cells (10 × 10⁶) were cultured with DMSO control or IDR E804 at concentrations of 50, 100, 200, and 400 nM for 48 hours. The IP of p-FLT3 receptor was performed as in Figure 1. Cells were washed, lysed, and subjected to 10% to 12% SDS-PAGE. Western blot analyses were detected with the indicated antibodies for the assessment of the expression level changes in STAT pathway molecules and Survivin, PARP, and cleaved PARP. Actin was used as a loading control. (C) MV4-11-R and MV4-11 cells were treated with various concentrations of ABT-869 alone or together with 2 nM IDR E804 for 48 hours. MTS assay was used to determine the viable cell number. Means are shown for 3 replicated experiments. (D) After parental MV4-11 cells were transiently transfected with pEGFP empty vector or pEGFP-STAT3 for 48 hours, RNA was extracted, followed by cDNA synthesis and relative quantification by RQ-PCR. The baseline expression of STAT3 and survivin in MV4-11 cells transfected with pEGFP vector was set as 1.0. The relative quantification of STAT3 in MV4-11 cells transfected with pEGFP-STAT3 was 354.6 ± 35 from 3 independent experiments. (E) ChIP assays were done using anti-STAT3 antibody or control anti-IgG antibody. PCR primers for the survivin gene promoter were applied to detect promoter fragment in immunoprecipitates. PCR controls included total sheared chromatin (total input), DNA isolated through the negative control IgG-ChIP, and no DNA at all (H₂O).