Extended Data Fig. 7: Extended Data Figure. 7 related to Fig. 5. Loss of G9 activity causes genome instability.

(a) Representative image of a combed DNA molecule labelled with CldU and IdU. This experiment was independently reproduced two times with similar results. (b) Bar chart showing the number of origin of replication per megabase (Mb) of DNA analyzed in the indicated conditions. (c) Bar chart showing the average inter-origin distance in kilobase (Kb) in the indicated conditions. (d) Plot showing the distribution of CldU track length in Kb in the indicated conditions. Mean ± SD of the track length distribution is shown. (n_WT= 55, n_G9aKO = 69, n_UNC0642 = 65 CldU tracks; **** = P ≤ 0.0001, ***= P ≤ 0.001, ns = non-significant, One-way ANOVA Kruskal-Wallis test followed by Dunn’s test). (a–d) This experiment was independently reproduced two times with similar results. (e) Top panel: Schematic of replication fork progression assay using CldU and IdU labeling. Bottom panel: CldU (red) and IdU (green) track length (µm) distribution for the indicated conditions. Mean ± SD of the track length distribution is shown.(n_UT= 158, n_UNC0642 = 163, n_roscovitin = 161, n_{UNC0642+roscovitin} = 165 CldU and IdU tracks analyzed; **** = P ≤ 0.0001, **= P ≤ 0.01, *= P ≤ 0.05, ns = non-significant, One-way ANOVA Kruskal-Wallis test followed by Dunn’s test). (f) Fork degradation assay using DNA fiber methodology. The distribution of the ratio of IdU to CldU track length (µm) was plotted for the given conditions. (n_UT= 155, n_UNC0642 = 152, n_roscovitin = 154, n_{UNC0642+roscovitin} = 154 tracks analyzed; **** = P ≤ 0.0001, ns = non-significant, One-way ANOVA Kruskal-Wallis test followed by Dunn’s test). (g, h) Representative images (top) and Quantification (bottom) of colony survival assay. Mean survival ± SD from 3 independent experiments in wild type (WT) and cells lacking G9a (G9a-/-) treated with different concentrations of olaparib (PARPi, g) or cisplatin (h) is shown. (**** = P ≤ 0.0001, ** = P ≤ 0.01, ns = non-significant, unpaired two-sided t-test). (i) Primpol was over-expressed in MRC-5 cells 48 h prior to the experiment and accumulation of ssDNA behind the replication forks upon primpol over-expression (primpol OE) and G9a inhibition (UNC0642) was assess using S1 nuclease. Right: IdU track length (µm) distribution for the indicated conditions. (n_{UT_S1-} = 120, n_{PrimpolOE_S1-} = 113, n_{UNC0642_S1-} = 120, n_{PrimpolOE+UNC0642_S1-} = 100, n_{UT_S1+} = 101, n_{PrimpolOE_S1+} = 110, n_{UNC0642_S1+} = 112, n_{PrimpolOE+UNC0642_S1+} = 114 tracks analyzed; **** = P ≤ 0.0001, *** = P ≤ 0.001, ** = P ≤ 0.01, * = P ≤ 0.05, ns = non-significant, Kruskal-Wallis test followed by Dunn’s test). Source numerical data are available in source data.

Source data