Fig 1: VLX600 reduces HR by inhibiting KDM histone demethylases. A, OVCAR-8-DR-GFP cells were transfected with individual siRNAs [negative control siLuc, positive control siBRCA1, or independent siRNAs targeting KDM4A (A#1, A#2), KDM4B (B#1, B#2), and KDM4C (C#1, C#2)], or cotransfected with a mixture of siRNAs targeting all three KDM4 family members (A#2, B#1, C#1). After 24 hours, cells were co-transfected with I-SceI plasmid and the same siRNAs as used in the first transfection, and incubated for 72 hours. Cells were then analyzed by immunoblotting (Supplementary Fig. S6B) and for GFP expression by flow cytometry. GFP expression is expressed as a percentage of the siLuc siRNA control. Mean ± SEM of at least three independent experiments. B, OVCAR-8 cells were transfected twice (24 hours between transfections) with individual siRNAs (negative control siLuc, positive control siBRCA1, siKDM4A #2, siKDM4B #1, siKDM4C #1) or were cotransfected with a mixture of KDM4 siRNAs (siKDM4A #2, siKDM4B #1, siKDM4C #1) and cultured for 48 hours. Cells were then trypsinized and immunoblotted (Supplementary Fig. S6C) or replated for clonogenic assay. Four hours after replating, cells were treated with olaparib and cultured for 8 to 10 days to allow colony formation. Colonies were manually counted. Mean ± SEM of three technical replicates from a single representative experiment of three independent experiments. C, OVCAR-8-DR-GFP cells were transfected with negative control siLuc, positive control siBRCA1, or a mixture of three KDM4 family member siRNAs (siKDM4A #2, siKDM4B #1, siKDM4C #1). After 24 hours, cells were cotransfected with I-SceI expression plasmid and the same siRNA as used in the first transfection, incubated for 18 hours, and analyzed by immunoblotting (Supplementary Fig. S6D) and by ChIP as in Fig. 3E. Mean ± SEM of three independent experiments. D and E, OVCAR-8 cells were transfected with negative control siLuc siRNA or co-transfected with a mixture of KDM4 siRNAs (siKDM4A #2, siKDM4B #1, siKDM4C #1) and cultured for 48 hours. Cells were then exposed to 2 Gy ionizing radiation, incubated for 6 hours, and then co-immunostained for RAD51 and γ-H2AX (D) or BRCA1 γ-H2AX (E). Colocalized foci were manually counted (B, C). Mean ± SEM of three independent experiments. F, OVCAR-8 and PEO14 cells were treated with 100 nmol/L or 150 nmol/L VLX600 continuously for 24 hours and histone extracts were immunoblotted for H3K9me3 and total histone H3. Immunoblot shown is representative of three independent experiments. F, OVCAR-8 and PEO14 cells were treated with 100 nmol/L or 150 nmol/L VLX600 for 24 hours and histone extracts were immunoblotted for H3K9me3 and total histone H3. Immunoblot shown is representative of three independent experiments. G, VLX600 increases H3K9me3 at I-SceI-induced DSBs. OVCAR-8-DR-GFP cells were transfected with an I-SceI expression plasmid. Two hours after plating, VLX600 was added, cells were cultured for 16 hours and then processed for ChIP as in Fig. 3E except that H3K9me3 antibody was used in place of RAD51 antibody. H–J, OVCAR-8 cells were transfected with negative control siLuc siRNA (H) or cotransfected with a mixture of KDM4 siRNAs (siKDM4A #2, siKDM4B #1, siKDM4C #1) (I) and cultured for 48 hours. Cells were then trypsinized and immunoblotted (Supplementary Fig. S6E) and subjected to colony-forming assays. For colony-forming assays, cells were plated, incubated 4 hours, and treated with indicated concentrations of olaparib with or without 40 nmol/L VLX600, and cultured for 8 to 10 days to allow colony formation. Colonies were manually counted. Mean ± SEM of three technical replicates from a single representative experiment of three independent experiments. (J) shows CI values calculated using the Chou–Talalay method in Calcusyn. Error bars, means ± SEM of three independent experiments. K, KDM4A was incubated with H3K9me3 peptide substrate using a succinate detection luminescent assay (IC50, ∼3.7 μmol/L). Mean ± SEM of three independent experiments. L, Proposed model of HR disruption by VLX600 (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001, unpaired Student t test).