Interactome rewiring following pharmacological targeting of BET bromodomains

U-2 OS cells were seeded onto 8-well LabTek II imaging chambers (ThermoFisher) in complete media with 1 μg/mL tetracycline and grown for 24 h to allow expression of GFP-tagged protein. Before imaging, the media was replaced with 200 μL of phenol red-free DMEM supplemented with 10% FBS, 1 mM sodium pyruvate, and 1× Glutamax (all Life Technologies). Images stacks were acquired on an imaging system (DeltaVision Elite, GE Healthcare). Cells were imaged at 37°C in 5% CO2 at 60×, 1.42NA, with 2×2 binning. Image Z-stacks of 24 μm were aquired at 2 μm intervals over 2 - 5 min as indicated. 20 s after the start of data acquisition, 100 μL of warm media containing 1.5 μM JQ1 was added to each cell chamber manually for a final concentration of 500 nM. The exposure time was 10 ms at 32% for GFP-tagged bait protein. Z-stacks were collected, deconvolved using softWoRx (v5.0, Applied Precision) and displayed as maximum intensity projections (pixel size 0.1075 μm). Images were cropped in ImageJ (National Institutes of Health). Supplemental Movie S1 – Flp-In T-REx U-2 OS cells expressing GFP-tagged BRD2 treated with 500 nM JQ1 for 2 min. Supplemental Movie S2 – Flp-In T-REx U-2 OS cells expressing GFP-tagged BRD3 (WT construct) treated with 500 nM JQ1 for 2 min. Supplemental Movie S3 – Flp-In T-REx U-2 OS cells expressing GFP-tagged BRD4 treated with 500 nM JQ1 for 2 min. Supplemental Movie S4 – Flp-In T-REx U-2 OS cells expressing GFP-tagged BRD3 BD1mut treated with 500 nM JQ1 for 2 min. Supplemental Movie S5 – Flp-In T-REx U-2 OS cells expressing GFP-tagged BRD3 BD2mut treated with 500 nM JQ1 for 2 min. Supplemental Movie S6 – Flp-In T-REx U-2 OS cells expressing GFP-tagged BRD3 (BD1:2)mut treated with 500 nM JQ1 for 2 min.将U-2 OS细胞接种于8孔LabTek II成像培养皿（ThermoFisher），使用含1 μg/mL四环素的完全培养基培养24小时，以诱导绿色荧光蛋白（GFP）标签蛋白的表达。成像前，将培养基更换为200 μL无酚红达尔伯克改良伊格尔培养基（DMEM），该培养基添加有10%胎牛血清（FBS）、1 mM丙酮酸钠及1× Glutamax（均购自Life Technologies）。图像序列采集于DeltaVision Elite成像系统（GE Healthcare）。成像过程维持37°C、5% CO₂的培养环境，采用60×物镜、1.42数值孔径（NA），并设置2×2像素合并模式。以2 μm为间隔采集总厚度24 μm的Z轴堆叠图像，采集时长为2至5分钟，依实验需求调整。数据采集开始20秒后，手动向每个培养孔加入100 μL预热培养基，其中含有1.5 μM JQ1，使最终药物浓度达到500 nM。针对GFP标签靶蛋白的曝光参数为：曝光时长10 ms，曝光强度32%。采集得到的Z轴堆叠图像经softWoRx（v5.0, Applied Precision）进行反卷积处理，随后以最大强度投影形式展示（像素尺寸为0.1075 μm）。图像裁剪操作使用ImageJ（美国国立卫生研究院）完成。 补充视频S1——表达GFP标签BRD2的Flp-In T-REx U-2 OS细胞经500 nM JQ1处理2分钟的成像视频。 补充视频S2——表达野生型GFP标签BRD3（WT构建体）的Flp-In T-REx U-2 OS细胞经500 nM JQ1处理2分钟的成像视频。 补充视频S3——表达GFP标签BRD4的Flp-In T-REx U-2 OS细胞经500 nM JQ1处理2分钟的成像视频。 补充视频S4——表达GFP标签BRD3 BD1突变体的Flp-In T-REx U-2 OS细胞经500 nM JQ1处理2分钟的成像视频。 补充视频S5——表达GFP标签BRD3 BD2突变体的Flp-In T-REx U-2 OS细胞经500 nM JQ1处理2分钟的成像视频。 补充视频S6——表达GFP标签BRD3（BD1:2）双突变体的Flp-In T-REx U-2 OS细胞经500 nM JQ1处理2分钟的成像视频。