Figure 2

ARV-825 sustained foci formation and BRD4 protein degradation longer than dBET1 in a proteasome-dependent manner. (a) 293A cells prepared as in Fig. 1 were treated with ARV-825 (0.01, 0.1 or 1 µM, left panels) or dBET1 (1, 5 or 25 µM, right panels) in 0.5% DMSO for the indicated time. Upper panels show the time course of foci formation until 24 h. Magnified areas of the early period highlighted with gray dashed boxes in upper panels are also shown in lower panels. The number of foci per cell was determined as in Fig. 1b. Data shown are the mean (n = 4) ± S.D. and are representative of three independent experiments. (b) 293A cells prepared as in Fig. 1 were treated with or without 2 µM MG132 in 0.1% DMSO for 1 h prior to the stimulation with 0.5% DMSO, 0.1 or 1 µM ARV-825, or 5 or 25 µM dBET1 for 0.5 or 6 h. The number of foci per cell was determined as in Fig. 1b. Data shown are the mean (n = 4) + S.D. and are representative of two independent experiments. *p < 0.001, **p < 0.0001. (c) 293A cells with knock-in of the HiBiT tag in the BRD4 gene at the N-terminus were cultured on a 384-well plate and stimulated with 0.1% DMSO, ARV-825 (0.1, 1, 10 or 100 nM, left panel) or dBET1 (1, 10, 100 or 1000 nM, right panel) for 3, 8 or 24 h. BRD4 expression levels were monitored with HiBiT-derived luminescent signals and normalized to the DNA contents in each well. The percentages of DMSO-treated cells are shown as the mean (n = 4) + S.D. Data shown are representative of two independent experiments. *p < 0.001.