Alpha Screen Assay
All reagents were diluted in 50 mM HEPES, 0.1 % BSA, pH 7.5 supplemented with 0.01 % Tween20 and allowed to equilibrate to room temperature prior to addition to plates. Catalytic turnover assays were run in 10µL volumes in 384-well plates at RT with enzyme (0.5-25nM), biotinylated substrate peptide (30-1000nM), Fe(II) (1-10µM), Ascorbate (100µM), 2OG (5-40µM). EDTA was used to quench the reaction (5µL) and AlphaScreen donor (Streptavidin-conjugated) and acceptor (ProteinA-conjugated) beads preincubated with peptide product antibodies were added (5µL). Plates were protected from light, incubated at room temperature for 60 minutes and read on a PHERAstar FS plate reader (BMG Labtech) using an AlphaScreen 680nm excitation/570nm emission filter set. IC50 values were calculated after normalization against corresponding DMSO controls.
Matrix-Assisted Laser Desorption/Ionisation-Time-of-flight (MALDI-TOF) Mass Spectrometry
KDM assays were carried out as reported [3] using an assay reaction consisting of JMJD2 (1 µM), Ferrous ammonium sulphate (10 µM), Ascorbate (100 µM), 2OG (10 µM), Histone H3 peptides (10 µM) in 50 mM HEPES (pH 7.5) with varying concentrations of inhibitors. The reaction was incubated at RT and 1:1 quenching with methanol followed by addition of four volumes of 20 mM triammonium citrate.
Cell-Based Assays
Flag-tagged JMJD2A was transiently overexpressed in HeLa cells either in the presence of a vehicle control (DMSO), 1 mM dimethyl-2,4-PDCA (a cell-permeable derivative of 2,4-PDCA), 2.5 mM dimethyloxalylglycine (DMOG, a cell-permeable derivative of N-oxalylglycine), or varying concentrations of IOX1. After 24 hours of incubation time cells were fixed and then analyzed by indirect immunofluorescence with Flag tag antibody to identify the cells overexpressing the demethylase and an antibody recognizing endogenous H3K9me3 to quantify the level of this histone modification. A series of images were collected for each treatment on a standard epifluorescence microscope and CellProfiler was used to analyze the images for DAPI signal thereby identifying the location of individual cells and create a boundary that delineates the volume of the nuclear compartment. As not all cells in a given field are transfected, the Flag-JMJD2A-expressing cells were identified by quantifying the immunofluorescence signal resulting from the Flag tag antibody staining and using the mock transfected cells as a baseline for the signal intensity of non-transfected cells. Once the transfected cells were identified, the nuclear H3K9me3 immunofluorescence signal for each cell was quantified by CellProfiler. The levels of H3K9me3 staining intensity were analyzed in the DMSO vehicle treated or inhibitor treated samples. As a control and a means of determining maximal possible inhibition of demethylase activity, cells expressing the JMJD2A H188A catalytically deficient mutant were also quantified in each experiment. The level of demethylase activity inhibition by IOX1 treatment was determined by quantifying the immunofluorescence signal from the DMSO treated sample (100% demethylase activity) compared to the maximal theoretical inhibition signal intensity as determined by the H3K9me3 signal in cells expressing the catalytically deficient JMJD2A H188A mutant (0% demethylase activity). For each treatment a minimum of 400 transfected cells was analyzed and the final values of inhibition were derived from inhibition experiments carried out on three separate days.
