Fig. 6: Inactivation of SETD2 and TP53 in neural stem cells leads to genome instability. | Nature Communications

Fig. 6: Inactivation of SETD2 and TP53 in neural stem cells leads to genome instability.

From: Multi-omic and single-cell profiling of chromothriptic medulloblastoma reveals genomic and transcriptomic consequences of genome instability

Fig. 6

a Inactivation of SETD2 in a p53 deficient background leads to the formation of micronuclei, aberrant nuclear structures linked with genome instability. Representative images based on three independent experiments are shown. Scale bar, 5 µm. b Quantification of micronuclei (n = three biological replicates; mean ± SD; p < 0.0001). c Inactivation of SETD2 leads to a larger nuclear area (n = three biological replicates; p < 0.0001). The bounds of the box represent the interquartile range (25th–75th percentile), the central line marks the median, and the whiskers extend to the minimum and maximum values. d, e Inactivation of SETD2 in a p53 deficient background leads to high levels of DNA double-strand breaks. Immunofluorescence analysis of γH2AX foci and quantification of γH2AX positive cells (Wild-type, n = five biological replicates; Non-target, n = three biological replicates; SETD2KO (#3, #8), n = four biological replicates; TP53KO, n = five biological replicates; TP53KO + SETD2KO (#3, #8), n = five biological replicates; mean ± SD; p < 0.0001). Scale bar, 5 µm. f, g Inactivation of SETD2 in a p53 deficient background leads to mitotic defects, as shown by immunofluorescence analysis of Phospho Histone H3 and Acetyl-α-Tubulin. Scale bar, 5 µm. h Inactivation of SETD2 in a p53 deficient background leads to increased proliferation rate. Metabolic activity results, indicating proliferation rate, are shown as absorbance values measured by MTT assay (Wild-type, n = four biological replicates; SETD2KO (#3, #8), n = five biological replicates; TP53KO, n = five biological replicates; TP53KO + SETD2KO (#3), n = three biological replicates; TP53KO + SETD2KO (#8), n = five biological replicates; mean ± SD; p = 0.0392). i, j Strand-seq analysis of wild-type and knock-out cells. Quantification of structural variants was performed with MosaiCatcher. Beta regression and Bonferroni–Holm method for multiple comparisons were used to test for statistical significance in b, e. One-way ANOVA and Bonferroni multiple comparison tests were used to test for statistical significance in c, h. Wald test on the inactivation status explaining counts of observed events in a negative binomial GLM (inactivation status and intercept) fit for each event type independently was used to assess significance in (j). Two sided p values are reported in (b, e, j). Source data are available as a Source Data file.

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