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Letters to the Editor
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Figure 3. MLL-partial tandem duplication drives the aberrant expression profile through BRD4. (A) Cell lysate of EOL-1 cells, either treated with vehicle (dimethyl sulfoxide [DMSO]) or with the indicated concentration of JQ1, were subjected to a chromatin immunoprecipitation (ChIP) assay. Antibodies against BRD4 or POL1RA (control) or an unspecific immunoglobulin G (IgG) were used for the pulldown. DNA of ADAMDEC1 and SLAMF8 were quantified using quantitative real- time polymerase chain reaction (qRT-PCR). The enriched binding of BRD4 to both genes, i.e., ADAMDEC1 and SLAMF8, was decreased after treatment of JQ1; **P<0.01, ***P<0.001. (B) Relative expression of ADAMDEC1 and SLAMF8 relative to GAPDH. qRT-PCR was performed on CD34+ selected cord blood (CB) cell samples (n=3, pooled) and three primary cell samples from MLL-partial tandem duplication (MLL-PTD) acute myeloid leukemia (AML) patients treated with JQ1 at 50 nM for 24 hours. Treatment with JQ1 leads to significantly lower expression of the genes in MLL-PTD patients’ cells but not in CB cells; ns = not sig- nificant, *P<0.05, **P<0.01, ***P<0.001. (C) Normalized expression of BRD4 downstream targets, BCL2, CDK6 and MYC, relative to GAPDH in EOL1 cells. Cells treated with JQ1 for 48 hours showed significantly reduced expression of the genes; *P<0.05, **P<0.01, ***P<0.001. (D) Western Blot analysis of EOL1 cells, which were previously treated with JQ1 or vehicle (DMSO) as control, validated the downregulation of these genes. Similar results were found when cells were transfected with a short hairpin RNA (shRNA) against MLL-PTD (shMLL-PTD) but not when transfected with a scramble control. Data of the densitometry are shown. RNA, cDNA, real-time PCR, ChIP and western blots were performed using previously published methods.13,16-18
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