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. 2017 Mar 16;24(3):371–380. doi: 10.1016/j.chembiol.2017.02.006

Figure 3.

Figure 3

KDOAM-25 Inhibits KDM5 Activity in Cells

(A) Half maximal effective concentration curves for KDOAM-25 against cells overexpressing FLAG-tagged KDM5B (left). Immunofluorescence assay showing inhibition of KDM5B (WT)-mediated H3K4me3 demethylation by KDOAM-25, but no effect of the negative control compound KDOAM-32. Immunofluorescence images showing HeLa cells stained with DAPI nuclear stain (blue), a specific antibody against H3K4me3 (green), and FLAG tag antibody (red) (right). Cells were transfected with KDM5B and treated with the indicated inhibitors. Arrowheads indicate cells transfected with KDM5B-FLAG.

(B) KDM5A and KDM5C are inhibited to a lesser extent by KDOAM-25 in cells overexpressing the respective demethylase. ****p ≤ 0.0001.

(C) KDOAM-25 has no effect on the histone substrates of the other KDM family members tested. No effect of any of the compounds is seen on any of the overexpressing catalytically inactive mutants, MUT; dotted lines in (A), (B), and (C). IF was read after 24 hr of compound treatment. Data represents the average and SE of at least 100 cells.