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. 2014 Dec 15;290(5):2744–2758. doi: 10.1074/jbc.M114.600759

FIGURE 9.

FIGURE 9.

Recruitment of BRD4 contributes to NUT-mediated transcriptional activation. A, U2OS 2-6-3 cells transfected with LacI-CHERRY-NUT were treated with 3 μm (−)-JQ1 or (+)-JQ1. 8 h after adding JQ1, cells were fixed and immunostained with p300, BRD4 C, Cdk9, Cyclin T1, or MED1 antibodies. The percentage of cells with these factors recruited to the transgene locus was calculated from more than 100 positively transfected cells. Values represent the mean ± S.D. of three independent experiments. B, U2OS 2-6-3 YFP-MS2 stable cells were transfected with LacI-CHERRY-NUT and treated with 3 μm (−)-JQ1 or (+)-JQ1. 8 h after adding JQ1, cells were fixed and subjected to single-cell image analysis of the YFP-MS2 intensity at the transgene array locus. The average YFP-MS2 intensity of the transgene array was quantified from more than 50 cells in each sample using ImageJ. Data represent the mean ± S.D. of all cells examined. C, U2OS 2-6-3 YFP-MS2 stable cells were treated as in B. The mRNA levels of the transgene reporter were measured by RT-qPCR and normalized to the LacI mRNA levels. Values represent the mean ± S.D. of three independent experiments. **, p < 0.01; ***, p < 0.001.