Extended Data Fig. 8: PML bodies colocalize with SP100-induced DNA damage.
a. Immunofluorescence in RPE1 SP110-KO cells stably expressing H2B-GFP stimulated with IFNβ staining for 53BP1 and PML. Scale bar, 20 µm. b. Individual IFNβ treated SP110-KO cell stained with 53BP1 (magenta) and PML (yellow) with blue profile line drawn across. Scale bar, 10 µm. c. Quantification of intensity across the blue profile line in b. d. Individual etoposide treated SP110-KO cell stained with 53BP1 (magenta) and PML (yellow) with blue profile line drawn across. Scale bar, 10 µm. e. Quantification of intensity across the blue profile line in d. f. Pearson object correlation of colocalization between PML and 53BP1 foci. Each data point represents one cell (n = 50 for each condition). Bars denote the median. A Mann-Whitney U test was used to determine statistical significance. g. Representative SP100 and PML immunofluorescence images in RPE1 SP110 KO cells treated with the indicated conditions. sgPML = CRISPRi knockdown of PML. Green arrows denote SP100 retained on mitotic DNA. Scale bar, 20 µm. h. Competition assay comparing ratio of RPE1 SP110 KO versus WT cells on day 7 with the indicated treatments. i. Competition assay comparing the ratio of RPE1 SP110-KO CRISPRi cells expressing the indicated sgRNA versus SP110 KO CRISPRi cells treated with IFNβ (n = 3 independent biological replicates). j. Knockdown verification of both PML targeting sgRNAs. Data points are technical replicates. Data is representative of n = 2 independent biological replicates. Bars in h-j denote mean ± s.d. Immunofluorescence experiments were performed n = 3 independent times with similar results. Source numerical data are available in source data.
