American Society of Hematology

Figure 5

$Figure 5. Role of HEXIM1 in the antiproliferative effect of I-BET151. (A) RQ-PCR analysis of MYC and HEXIM1 mRNA expression in H929 and KMS12BM myeloma cells over 24 hours after treatment with I-BET151 (1 μM) or DMSO control. Expression is normalized against the RNA levels at t = 0. Data shown are representative of 2 independent experiments; mean ± SEM of technical triplicate assays for each time point is shown. (B) HEXIM1 protein levels in H929 myeloma cells as assessed by immunoblotting after treatment with I-BET151 1 μM (+) or DMSO (−) control at the time points indicated. Two immunoblots with different time points are shown. (C) Relative levels of active (HEXIM1-free) and inactive (HEXIM1-bound) CDK9 upon treatment with I-BET151 (1 μM) of H929 cells. Following differential salt extraction of nuclei and running of low- (inactive CDK9 fraction) and high-salt (active CDK9 fraction) extracts for each time point side-by-side on sodium dodecyl sulfate polyacrylamide gel electrophoresis, the same membrane was immunoblotted with anti-HEXIM1 and CDK9 antibody. (D). ChIP assay performed under the same conditions described in Figure 4, showing occupancy at HEXIM1 promoter of BRD2-4 at 0, 2, and 4 hours after treatment with I-BET151 (1 μM; n = 3). I, inactive CDK9; A, active CDK9.$

Role of HEXIM1 in the antiproliferative effect of I-BET151. (A) RQ-PCR analysis of MYC and HEXIM1 mRNA expression in H929 and KMS12BM myeloma cells over 24 hours after treatment with I-BET151 (1 μM) or DMSO control. Expression is normalized against the RNA levels at t = 0. Data shown are representative of 2 independent experiments; mean ± SEM of technical triplicate assays for each time point is shown. (B) HEXIM1 protein levels in H929 myeloma cells as assessed by immunoblotting after treatment with I-BET151 1 μM (+) or DMSO (−) control at the time points indicated. Two immunoblots with different time points are shown. (C) Relative levels of active (HEXIM1-free) and inactive (HEXIM1-bound) CDK9 upon treatment with I-BET151 (1 μM) of H929 cells. Following differential salt extraction of nuclei and running of low- (inactive CDK9 fraction) and high-salt (active CDK9 fraction) extracts for each time point side-by-side on sodium dodecyl sulfate polyacrylamide gel electrophoresis, the same membrane was immunoblotted with anti-HEXIM1 and CDK9 antibody. (D). ChIP assay performed under the same conditions described in Figure 4, showing occupancy at HEXIM1 promoter of BRD2-4 at 0, 2, and 4 hours after treatment with I-BET151 (1 μM; n = 3). I, inactive CDK9; A, active CDK9.

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