Fig. 3: Loss of PBK results in reduced mitotic chromosome compaction and increased retention of specific factors.
a Mitotic chromosomes from demecolcine-arrested Pbk+/+ and Pbk–/– mouse preB cells visualised by flow cytometry after staining with Hoechst 33258 and Chromomycin A3. Gates for the purification of chromosomes 3 and 19 are shown, representative of three replicates. b Flow-purified mitotic chromosomes 3 (upper) and 19 (lower) from arrested Pbk+/+ and Pbk–/– mouse preB cells, cytocentrifuged onto slides and stained with DAPI. Representative of chromosomes from three independent replicates; scale bar = 5 μm. c Size quantification of mitotic chromosomes 3 and 19, flow-purified from arrested Pbk+/+ and Pbk–/– mouse preB cells, measuring total (left) and DAPI-dense centromeric (right) areas. Quantification from one experiment, representative of three independent replicates (chromosome 3: n = 117, 65 (total area) and n = 96, 49 (centromeres); chromosome 19: n = 133, 189 (total area) and n = 113, 164 (centromeres); boxplots show median, interquartile range and Tukey whiskers; ****p < 0.0001, unpaired two-tailed t tests). d Size quantification of chromosomes 3 and 19 in metaphase spreads from arrested Pbk+/+ and Pbk–/– mouse preB cells, identified by chromosome paints. Quantification from one experiment, representative of two replicates (n = 26, 39 (chr 3) and n = 24, 39 (chr 19); plots show median and interquartile range; **p = 0.0017, *p = 0.0172, unpaired two-tailed t tests). e Volcano plots highlighting specific factors enriched on or depleted from Pbk+/+ and Pbk–/– mitotic chromosomes, compared to pre-sorted lysate pellets (green = enriched, blue = depleted, dark grey = not significantly enriched/depleted; modified two-tailed t test with permutation-based false discovery rate (FDR) < 0.05, S0 = 0.1; n = 4 chromosome samples and n = 3 lysate pellet samples; only selected annotation labels are shown for proteasome and C2H2-zinc finger proteins for clarity; volcano plots highlighting all enriched/depleted factors are shown in Supplementary Fig. 3f). Source data for Fig. 3c, d are provided as a Source Data file.
