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Fig. 7 | Journal of Hematology & Oncology

Fig. 7

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. 7

The PROTAC/BRD4 degrader, ARV-825, upregulates MICA expression in SKO-007(J3) cells. a Treatment of SKO-007(J3) cells with ARV-825 induced a strong degradation of BRD4 that was reverted in the presence of lenalidomide. Lysates of SKO-007(J3) cells, untreated or treated with ARV-825 (0.2 μM), lenalidomide (10 μM), or the combination of the two drugs for 24 h, were subjected to Western blotting using anti-BRD4 or anti-p85 antibodies. b ARV-825 inhibits the expression of IRF4. Lysates of SKO-007(J3) cells, untreated or treated with the indicated (μM) concentrations of ARV-825 for 48 h, were subjected to Western blotting using anti-IRF4 or anti-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. c, d ARV-825 inhibits mRNA expression of IRF4 and cMYC. Real-time PCR analysis of total mRNA obtained from SKO-007(J3) cells, untreated or treated with the indicated (μM) concentrations of ARV-825 for 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). e, f MICA, MICB, and PVR/155 cell surface expression were analyzed by flow cytometry on SKO-007(J3) cells treated with the indicated concentrations of ARV-825 for 72 h. The MFI of MICA, MICB, and PVR/155 were calculated based on at least four independent experiments and evaluated by paired Student t test (*P < 0.05). In e, a representative histogram of MICA upregulation is shown. The grey-colored histograms represent basal expression of the indicated ligand, while thick black histograms represent the expression after treatment with the drug. Data are representative of one out of four independent experiments. g ARV-825 increases mRNA expression of MICA in SKO-007(J3) cells. Real-time PCR analysis of total mRNA obtained from SKO-007(J3) cells, untreated or treated with the indicated (μM) concentrations of ARV-825 for 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). h MICA cell surface expression was analyzed by flow cytometry on patient-derived MM cells treated with the indicated ARV-825 for 72 h. The grey-colored histograms represent basal expression, while thick black histograms represent the expression after treatment with the drug

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