Fig. 5

NPM1 KD triggers the silencing of DNA repeats and disrupts the spatial organization of peri-nucleolar heterochromatin. A Representative immunoblot analysis of the levels of the H3K27me3 and NPM1 upon 3 days of NPM1 KD in N2a cells. Ponceau staining was used for total protein normalization. B Quantification of the signal intensity of H3K27me3 shown in A was done using Fiji (ImageJ). H3K27me3 levels were first normalized to the corresponding Ponceau signal and then presented as a ratio over the control values. Statistical analysis was performed on n = 5 biological samples using an unpaired two tailed t-test. *p < 0.05, **p < 0.01. Error bars represent S.D. C Individual data points plot showing the fold change enrichment of H3K27me3 calculated after ChIP-qPCR upon NPM1 KD (KD/CTR, 3 days). gSAT (gamma satellites), mSAT (major satellites). Statistical analysis was performed on n = 4 biological replicates using an unpaired two tailed t-test. *p < 0.05, **p < 0.01. Error bars represent S.D. D Individual data points plot showing the fold change (NPM1 KD/CTR) in the expression of the indicated DNA repeats calculated by RT-qPCR upon 3 days of NPM1 KD in N2a cells. Gapdh was used as reference gene for normalization. Abbreviations as in C. Statistical analysis was performed on n = 6 biological replicates using an unpaired two tailed t-test; *p < 0.05, **p < 0.01. Outliers were identified using a Grubbs method. Error bars represent S.D. E Confocal immunofluorescence images of immune-FISH performed on N2a cells after 3 days of NPM1 KD using cy3-labeled probes specific for mSat (red). GFP antibody (green) was used to highlight NPM1 KD cells. Scale bars 10μm. F Quantification of the average number of mSat foci in control (CTR) and after 3 days of NPM1 KD. Statistical analysis was performed on n = 3 biological replicates and on n = 68 (CTR) and n = 68 (KD) cells using a two-way ANOVA with Sidak’s post-hoc test. *p < 0.05, **p < 0.01. Error bars represent S.D. G Quantification of the mean area of mSat foci per cell in control (CTR) and after 3 days of NPM1 KD. Statistical analysis was performed on n = 3 biological replicates and n = 20 nuclei per condition using a two-way ANOVA with Sidak’s post-hoc test. *p < 0.05, **p < 0.01.Error bars represent S.D. H Nuclei were divided in 3 categories according to the average size of mSat foci per nuclei: small (0.2–1 μm²), medium (1–2 μm²) and large (> 2 μm²). Statistical analysis was done between classes using a two-way ANOVA with Sidak’s post-hoc test. *p < 0.05, **p < 0.01. Only small and large mSat foci show a significant difference in their average size between control (CTR) and NPM1 KD. I Individual data points plot showing the expression of the indicated DNA repeats upon NPM1 depletion (KD), EPZ treatment (EPZ) or EPZ treatment of NPM1 KD (KD + EPZ) in N2a cells. Gapdh was used as reference gene for normalization. Statistical analysis was performed on n = 6 biological replicates using an unpaired two tailed Student's t-test; *p < 0.05, **p < 0.01. Error bars represent S.D