Fig. 2: KDM4B^−/− mouse ES cells show replication stress.

a Protein immunoprecipitation with an anti-Flag antibody in ES cells expressing HA-H3.3 and either Flag-tagged KDM4A, KDM4B or KDM4C, followed by western blot analysis with an anti-HA antibody. b ChIP-sequencing analysis of KDM4 -A³⁴, -B³³ and -C³³ with input sequencing. Data shows reads which aligned to telomeres, normalised for total read counts showing KDM4B binding to telomeres. c ChIP-qPCR analysis of KDM4B at telomeres in cell cycle-synchronised cells (n = 3 independent experiments). d Western blot analyses of WT and KDM4B cells using antibodies against H3K9me3, H3K36me3, H3 and ACTIN. e ChIP-qPCR analyses of H3.3, total H3, H3K9me3, ATRX, HP1α. Level of H3K9me3 was normalised to total H3 levels (H3K9me3/H3) (n = 3 independent experiments). f ChIP-qPCR analyses of BrdU incorporation at telomeres and Gapdh promoter in WT and Kdm4b^−/− cells (n = 4 independent experiments). g, h Immunofluorescence analyses of TERF1 (red; diluted at 1/500) and γH2AX (green; diluted at 1/1500) in WT and Kdm4b^−/− cells with and without 1 mM APH treatment for 5 h. Arrows indicate presence of DNA damage (γH2AX) at telomeres (TERF1). Scale bars: 5 μm. Percentages of co-localised TERF1 and γH2AX foci in two independent Kdm4b^−/− cell lines (Kdm4b^−/− #1 and #2) are shown in h. For each cell line, >1500 telomeric or TERF1 foci were counted (n = 4 independent experiments). Percentage of co-localised TERF1/γH2AX foci are determined as percentages of telomeric foci that co-stained with γH2AX over the total number of telomeric foci counted. c, e, f, h Data are presented as mean values ± SD. *indicates p < 0.05 and **indicates p < 0.005, Student’s t-test with two-tailed distribution. a, d, g Representative images and blots from at least n = 3 independent experiments. Source data are provided with this paper.