Interactome rewiring following pharmacological targeting of BET bromodomains
Description
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.