Extended Data Fig. 6: SP100 induces mitotic genotoxicity.
a,b. 53BP1 immunofluorescence in RPE1 TP53 +/+ (a) WT and (b) SP110;SP100 dual-KO cells stably expressing H2B-GFP treated with buffer, IFNβ (10 ng/mL), or etoposide (ETO; 25 µM) for 72 h. Scale bar, 20 µm. c. Western blot of the lysates from the indicated RPE1 TP53+/+ genotypes treated with buffer (mock), ETO, IFNβ, or transduced with SP100 cDNA. Note the transduction efficiency was ~30% and cells were not selected for positive integrations. Lysates were probed for phosphorylated KAP1 (S824) and cyclin B1 with GAPDH serving as the loading control. d. Representative fluorescence images of catalytically inactivate GFP-RNaseH1D210N in U2OS cells treated with the indicated siRNAs (NT = non-target). Scale bar, 50 µm. Results are quantified in Fig. 3f. e. Quantification of knockdown efficiencies of the labelled genes with the indicated siRNAs. Each data point represents a technical replicate. Results are representative from n = 2 experiments. f. RT-qPCR of CDKN1A (p21) in untreated and IFNβ treated cells of the indicated genotype. Each data point corresponds to technical replicates from a representative experiment performed n = 2 times. g. Immunofluorescence of human centromere in RPE1 TP53+/+; SP110−/− cells treated with IFNβ. Scale bar, 10 µm. h. Western blots of cellular fractions of G2/M arrested cells to identify SP100 localization. So = soluble/cytoplasmic fraction (marked by GAPDH); Nu = nuclear soluble fraction (marked by ZEB1); Ch = chromatin fraction (marked by histone H2B). Bars in e and f denote mean ± s.d. Images in a, b and g are representative from n = 3 independent biological replicates. Western blots in c and h were performed 2 independent times with similar results. Source numerical data and unprocessed blots are available in source data.
