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Fig. 4 | Journal of Hematology & Oncology

Fig. 4

From: Inhibition of bromodomain and extra-terminal (BET) proteins increases NKG2D ligand MICA expression and sensitivity to NK cell-mediated cytotoxicity in multiple myeloma cells: role of cMYC-IRF4-miR-125b interplay

Fig. 4

MICA promoter activity is enhanced by BETi in MM cells. a SKO-007(J3) cells were cotransfected with 3 μg of the indicated luciferase reporter vector + 1 μg of pRL-TK expression vector as described in the “Methods” section. Four hours after transfection, cells were left untreated (−) or were stimulated with 0.5 μM JQ1 for 48 h. Cells were then harvested and protein extracts were prepared for the luciferase and Renilla assays. Results are expressed as relative luciferase activity normalized to protein concentration as well as to Renilla activity produced off the internal control plasmid and represent the mean value of four independent experiments (*P < 0.05). b–e BETi represses cMYC and IRF4 mRNA expression in SKO-007(J3) MM cells. Real-time PCR analysis of total mRNA obtained from SKO-007(J3) cells, untreated or treated with the indicated BETi as described above for 24 and 48 h. Data, expressed as fold change units, were normalized with GAPDH and referred to the untreated cells considered as calibrator and represent the mean of three experiments (*P < 0.05). f Lysates of SKO-007(J3) cells untreated or treated with BETi for 24 or 48 h were subjected to Western blotting using anti-IRF4 and actin antibodies. The proteins transferred to nitrocellulose membranes were stained with Ponceau to verify that similar amounts of proteins had been loaded in each lane. Data are representative of one out of three independent experiments

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