(a) E-isomer lowers H3K9me3 demethylase activity in cancer cells dose-dependently. Activity is inhibited at lower doses of JIB-04 over prolonged exposure. Error bars represent s.e.m. across 2–3 independent assays. Asterisks denote statistically significant decreased activity of E-isomer versus DMSO-treated cells with P<0.05 for one-tailed t-tests. Cells were plated at five-times higher density than for standard viability assays to ensure sufficient surviving cells/enzymatic activity post drug treatment. (b) Knockdown of JARID1B (left panel) reduces cancer cell viability (middle panel) and increases c10orf10 expression (right panel), as does 4 h JIB-04 treatment (see Supplementary Fig. S4). Error bars represent experimental error (left and right panels) or s.d. (middle panel) from the average of triplicates. (c) H3K4me3 levels are increased at the c10orf10 promoter in HCC4017 cells in response to treatment with 0.5 μM JIB-04 E-isomer for 24 h as measured by chromatin immunoprecipitation. (d) The increased basal viability of cells transiently overexpressing JMJD2B, but not the H189/E191Q JMJD2B mutant (mut), compared with mock-transfected cells (mock), is shown on the left panel. Overexpression of JMJD2B but not its mutant in H2009 lung cancer cells right shifts the response to JIB-04 (right panel). Averages across eight replicates are shown+s.d. The middle panel shows the expression levels of the HA-tagged wild type and mutant constructs for the cells in the right panel.