Fig. 2

Immunofluorescence assay assessing and comparing potencies of inhibitors in cells. a Widefield fluorescence imaging of HeLa cells after dosing with inhibitor, fixing and staining with i, iv, vii DAPI (blue), ii, v, viii histone antibody for H3K4me3 (green), and iii, vi, ix a FLAG-tag antibody that demarcates cells overexpressing KDM5B (red). Cells overexpressing the wild-type (WT) KDM were treated with the inhibitor KDOAM-21 (i–iii) or with DMSO (iv–vi); controls cells overexpressing an inactive mutant (MUT) were treated with DMSO (vii–ix). Arrowheads indicate KDM overexpressing cells. The scalebar represents 50 µm, b–d measurement of the average histone mark intensity in the transfected HeLa cells allows quantification of inhibitor potency against each target. KDOAM-21 (red), KDOAM-20 (blue) and the inactive control KDOAM-32 (black) were tested on multiple targets, b illustrates the characteristic dose–response curves of the potent KDOAM-21, weak KDOAM-20 and inactive KDOAM-32 compounds on KDM5 family members. The wild type (WT) and catalytically inactive mutant (MUT) of each target are shown as bold and dashed lines, respectively. The baseline for the mutant is of similar intensity as the nontransfected cells and approximately represents complete inhibition of the target demethylase activity, c selectivity of KDOAM-20 and KDOAM-21 against KDM5 is further demonstrated in cells by their weaker potency against other demethylases, i.e. members of the KDM3, KDM4 and KDM6 families. In d, KDOAM-21 and, to a lesser extent, KDOAM-20 are shown to have activity against endogenous KDMs as seen by changes in H3K4me3 in nontransfected cells. KDOAM-32 retains its inactivity