Fig. 4

Pharmacological proof-of-concept for the in situ cell extraction methodology using reference BET bromodomain inhibitors. a Outline of the in situ cell extraction procedure. b 96-well plate view for BET bromodomain inhibitor treatment of HeLa cells expressing FLAG-tagged full-length BRD4. Cells were subjected to in situ cell extraction and two-color IF staining for TRIM24 (green) and histone H3 (red). The plate layout shows serial dilution of 3 BET inhibitors (JQ1, RVX-280 and I-BET) including wells with DMSO and secondary antibody controls. c Magnified IF images show decrease of BRD4 staining upon JQ-1 treatment (10 µM), while histone H3 (red) and Hoechst staining remains unchanged. d Quantification of drug dose responses based on IF image analysis. e Image analysis sequence using Harmony software. Left panel cell segmentation and gating strategy based on Hoechst (blue) staining eliminating cells of inappropriate morphology (red on the second image). Right panel the same cells with co-staining for BRD4 (green) and H3 (red). The mean intensity of the BRD4 signal was measured per nucleus and the average nucleus intensity was calculated from about 1000 gated cells (i.e., excluding the masked white cells on the fourth image)