Treatment with BET antagonist reduces nuclear expression of RelA and BTK, and depletes mRNA expression of NF-kB target genes in MCL cells. (A) Confocal immunofluorescence analysis of RelA expression and cellular localization in Mino cells following treatment with JQ1 for 24 hours. Original magnification, ×63. The bar graph (right) shows quantification of the fluorescein isothiocyanate signal intensity of the JQ1-treated Mino cells relative to the untreated cells. (B) Mino cells were treated with the indicated concentrations of JQ1 for 24 hours (top). Following this, nuclear and cytoplasmic fractions were prepared and immunoblot analyses were conducted for BTK and RelA. The localization of Lamin B served as a fraction and loading control. The graph (bottom) shows the relative expression of p-BTK and BTK in the nucleus, determined by densitometry and normalized against the expression of Lamin B. Representative immunoblots are shown. (C) qPCR performed on cDNA from Mino cells treated with the indicated concentrations of JQ1 for 8 hours. Relative expression of each target was normalized against GAPDH. (D) Immunoblot analyses were conducted on the lysates of MO2058 (left) and Mino (right) cells treated with JQ1 for 24 hours, as indicated. The numbers beneath the bands represent densitometry analysis performed on the blots and normalized to the β-actin loading control. Cyto, cytoplasmic; DAPI, 4′,6 diamidino-2-phenylindole; Nuc, nuclear.