Fig. 3: The inhibition of KDM4B by H3.3^G34R contributes to ALT activation.

a, b Telomere length analyses of Kdm4b^−/−; Tp53^−/−; Tert^−/− and Kdm4b^−/−APT-tKO cells over a 6-month period following knockout of Kdm4b in Tp53^−/−; Tert^−/− (TP) and APT-tKO cells (n = 3 independent experiments). c, d Immunostaining of APBs in Kdm4b^−/−; Tp53^−/−; Tert^−/− and Kdm4b^−/−APT-tKO cells, shown by co-staining of TERF1 (green) and PML (red). Arrows indicate co-staining of TERF1 and PML. Percentages of co-localised TERF1 and PML foci in Kdm4b^−/−; Tp53^−/−; Tert^−/− #1 and Kdm4b^−/−APT-tKO #1 are shown in d (n = 5 independent experiments; >1200 foci counted for each line). e ChIP-PCR analyses of KDM4B, H3K9me3 and H3 in WT, APT-tKO and APT-tKO^12m Kdm4b^−/− cells. Level of H3K9me3 was normalised to total H3 levels (H3K9me3/H3) (n = 3 independent experiments). f, g Immunostaining of TERF1 (green; diluted at 1/500) and HP1α (red; diluted at 1/500) in WT, APT-tKO^12m and APT-tKO^12m Kdm4b^−/− cells, 4 months in culture following Kdm4b knockout. Arrows indicate co-staining of TERF1 and HP1α. Percentages of co-localised TERF1 and HP1α foci in APT-tKO^12m, APT-tKO^12m Kdm4b^−/−#1 and #2 are shown in g. For each cell line, >1500 telomeric or TERF1 foci were counted (n = 3 independent experiments). Percentage of co-localised TERF1/HP1α foci are determined as percentages of telomeric foci that co-stained with HP1α over the total number of telomeric foci counted. a, b, d, e, g Data are presented as mean values ± SD. *indicates p < 0.05 and ** indicates p < 0.005, Student’s t-test with two-tailed distribution. c, f Scale bars: 5 μm; representative images from at least n = 3 independent experiments. Source data are provided with this paper.