Figure 5.

Npm2 levels correlate with nuclear histone levels and altered chromatin topology. (A) Nuclei assembled in X. laevis egg extract for 30, 60, and 90 min were stained with an H2B antibody and Hoechst. Total nuclear H2B staining intensities were measured for ≥60 nuclei per condition and normalized to the 30-min time point. For each nucleus, two Hoechst intensity line scans were acquired through the middle of the nucleus. The SD of all intensity values along each line was calculated and normalized to the average intensity to obtain a value we term the chromatin heterogeneity index. Larger values correspond to a more heterogeneous chromatin distribution. 54–68 line scans were quantified per condition (60 on average). (B) Different stage nuclei were stained with an H2B antibody and Hoechst. Total nuclear H2B staining intensities were quantified for ≥100 nuclei per stage and normalized to stage 12. For chromatin heterogeneity indexes, 22–79 line scans were quantified per condition (45 on average). (C) Microinjection experiments were performed as in Fig. 4 A. Isolated nuclei were stained with an H2B antibody and Hoechst. Total nuclear H2B staining intensities were measured for ≥97 nuclei per condition and normalized to the XB-microinjected controls. For chromatin heterogeneity indexes, 34–52 line scans were quantified per condition (41 on average). Data from four independent experiments are shown. Two-tailed Student’s t tests assuming equal variances; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ns, not significant. Error bars represent SD.