Fig. 3

ID4 interacts with DNA damage proteins at fragile chromatin sites in a DNA damage-dependent manner. a Representative proximity ligation assay (PLA) confocal images of ID4 and MDC1 interactions in MDA-MB-468 cells. siRNAs targeting MDC1 (siMDC1) and a scrambled control (siControl) used to show specificity of the assay. PLA foci (green), DAPI (blue) and phalloidin (magenta). 20 μm scale. b Graph showing quantification of PLA interactions between ID4 and MDC1 in cells treated with siControl and siMDC1. Data shown in comparison to the interaction between ID4:V5 and ID4:FLAG treated with siControl. 50–100 cells captured per condition. Quantification of interactions (number of dots/ cell). One-way ANOVA, multiple comparisons test. ****p < 0.0001. Error bars represent standard deviation. c ChIP-qPCR analysis of ID4 binding in untreated HCC70 cells compared to cells treated with ionising radiation (5 Gy with 5 h recovery time prior to fixation). ID4 binding normalised to negative control region, input DNA and IgG control. Data for each gene region is shown for both untreated (left) and ionising radiation-treated (right) cells, connected by a line. Representative of two independent experiments. Quantification of a time course of d γH2AX and e 53BP1 DNA damage foci formation following ionising radiation. ID4 was depleted from cells using the SMARTChoice inducible shRNA system following treatment with doxycycline for 72 h prior to treatment with 5 Gy ionising radiation at time 0 and allowed to recover for 0.25 and 8 h prior to analysis. Four to five images were taken for each condition; 100–200 cells in total. Number of foci per cell nucleus was calculated using FIJI by ImageJ (Schindelin et al., 2012), and samples were then collated and analysed using the Pandas package in Python 3.5. Data is normalised to 0 h, no DOX, no IR time point. n = 3–5, Student’s t test, *p < 0.05. Error bars represent standard error