Supplementary Figure 6: Life cell imaging, super resolution and DNA repair assays data.
From: XLF and APLF bind Ku80 at two remote sites to ensure DNA repair by non-homologous end joining
(a-b) Dynamics of CFP-(A-KBM) (a) and (X-KBM) (b) at laser damaged sites in U2OS cells expressing wild-type (WT), E133M or Q162E mutant Ku80 as in Fig. 3 b). Mean values of the relative fluorescence with SEM were calculated from 20, 23 and 22 independent measurements for A-KBM with WT, E133M or Q162E mutant Ku80 in (a) and from 48, 29 and 29 independent measurements for X-KBM with WT, E133M or Q162E mutant Ku80 in (b), respectively. p values at last time point: (a) WT vs E133M p = 0.831; WT vs Q162E p = 0.59519; (b) WT vs E133M p = 0.0003; WT vs Q162E p = 0.0111. (c-d) Dynamics of wild-type (WT) and mutant CFP-Ku80 at laser damaged sites in U2OS cells. Mean values of the relative fluorescence with SEM were calculated from 25, 24, 20 and 15 independent measurements for WT, I112R, E133M and Q162E mutant Ku80 in (c) and from 25 and 26 independent measurements for WT or I112R/E133M mutant Ku80 in (d), respectively. p values at last time point: (c) WT vs Q162E = 0.9252; WT vs I112R p = 0.2734; WT vs E133M p = 0.1101. (d) WT vs I112R-E1333M p = 0.5362. (e-f) Analysis of XLF foci in U2OS cells by super-resolution. (e) Statistics of XLF foci size: each plot represents the average XLF foci size (indicated as radius translated from the correlation radius) in one nucleus. Box’s height displays the standard deviation with the mean value labelled in the middle. 87, 110, 64, and 79 nuclei were taken in account for WT, E133M, I112R, and E133M-I112R double-mutant, respectively. The p-values were obtained by the t-test; (f) Statistics of the Cross-Pair-Correlation between Ku and XLF: Ku and XLF were stained with antibodies labelled by different fluorophores (Alexa488 conjugated rabbit anti-Ku80, abcam198586, Alexa647 conjugated goat anti-mouse secondary + Mouse anti-XLF, NBP2-03275), and dual-colour super-resolution imaging was performed to examine the cross-correlation between Ku and XLF foci within each nucleus. Each plot represents the cross-correlation amplitude calculated across one nucleus. Box’s height displays the standard deviation with the mean value labelled in the middle. 83, 107, 57, and 72 nuclei were taken in account for WT, E133M, I112R, and E133M-I112R mutants respectively. The p-value were obtained by the t-test.
