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.

U-2 OS 细胞于 8 孔 LabTek II 显微镜培养室（赛默飞世尔）中接种，置于全培养基中，加入 1 μg/mL 四环素，培养 24 小时以允许 GFP 标记蛋白的表达。成像前，培养基被更换为 200 μL 无酚红 DMEM 培养基，并补充 10% FBS、1 mM 丙酮酸钠和 1× Glutamax（所有均为 Life Technologies 产品）。图像堆栈在 DeltaVision Elite 显微成像系统（通用电气医疗保健）上获取。细胞在 37°C、5% CO2 条件下，以 60×、1.42NA 焦距和 2×2 合并的方式进行成像。在 2-5 分钟的间隔内，以 2 μm 的步长获取了 24 μm 的图像 Z 堆栈。数据采集开始后 20 秒，手动向每个细胞培养室添加含 1.5 μM JQ1 的 100 μL 温培养基，以实现最终浓度 500 nM。GFP 标记诱饵蛋白的曝光时间为 10 ms，光圈设置为 32%。Z 堆栈通过 softWoRx（v5.0，Applied Precision）进行去卷积处理，并以最大强度投影（像素大小 0.1075 μm）的形式显示。图像在 ImageJ（美国国立卫生研究院）中进行裁剪。

补充视频 S1 – 表达 GFP 标记 BRD2 的 Flp-In T-REx U-2 OS 细胞经 500 nM JQ1 处理 2 分钟。

补充视频 S2 – 表达 GFP 标记 BRD3（野生型结构）的 Flp-In T-REx U-2 OS 细胞经 500 nM JQ1 处理 2 分钟。

补充视频 S3 – 表达 GFP 标记 BRD4 的 Flp-In T-REx U-2 OS 细胞经 500 nM JQ1 处理 2 分钟。

补充视频 S4 – 表达 GFP 标记 BRD3 BD1mut 的 Flp-In T-REx U-2 OS 细胞经 500 nM JQ1 处理 2 分钟。

补充视频 S5 – 表达 GFP 标记 BRD3 BD2mut 的 Flp-In T-REx U-2 OS 细胞经 500 nM JQ1 处理 2 分钟。

补充视频 S6 – 表达 GFP 标记 BRD3（BD1:2）mut 的 Flp-In T-REx U-2 OS 细胞经 500 nM JQ1 处理 2 分钟。