Fig. 2
Characterization of engineered HeLa cells expressing TRIM24 full-length or isolated PHD-bromodomain. a Western blot analysis of stable HeLa cell lines expressing either vector control (−) or dual-tagged (FLAG-TRIM24-V5) full-length (FL) or isolated PHD-bromodomain (PB). Lysate from cells treated with Suberoylanilide Hydroxamic Acid (SAHA) for 2 h (2 µM) or vehicle control (DMSO) were probed with the indicated antibodies confirming SAHA-induced acetylation of H3K23. b Immunoprecipitation of endogenous full-length TRIM24 (using anti-TRIM24 antibody) under non-treated (DMSO) and SAHA-treated (2 µM, 2 h) conditions. c Immunoprecipitation of the TRIM24 PHD-bromodomain (using anti-FLAG beads) and looking at co-immunoprecipitated histone H3 in TRIM24-PB cells non-treated or treated (2 µM, 2 h) with either Trichostatin A (TSA) or (SAHA). d Proximity ligation assay (PLA) of TRIM24-PB cells using the goat anti-V5 (i.e., TRIM24-PB) and mouse anti-histone H3 antibody pair, showing an increase in co-localization of TRIM24 with histone H3 in response to SAHA treatment (2 µM, 2 h). The increase in proximity of the two proteins (number of blue PLA dots) is quantified in Additional file 1: Figure S2. e Representative IF images of TRIM24-PB cells following SAHA treatment (2 µM, 2 h) showing acetylation of histone H3K23 (yellow) and increased chromatin binding of TRIM24-PB (red) with Hoechst nuclear staining (blue) serving as a control for the in situ cell extraction procedure. f SAHA dose–response and time-course studies. Left Graph IF quantification (Arbitrary Units) of H3K23Ac in Hela control and TRIM24-PB cells upon SAHA treatment (2 h). Right Graph IF quantification (average IF signal per nucleus for TRIM24-PB and H3K23Ac as percent of the non-SAHA-treated control (PoC)) under in situ cell extraction (+) and non-extraction (−) conditions. g Schematic diagram for measuring the dual-tagged TRIM24 protein in cell lysates (Top) showing AlphaLISA results for HeLa cells (Parental, TRIM24-FL and TRIM24-PB) using anti-FLAG donor and anti-V5 acceptor beads (Middle) compared to immunoblotting (Bottom)