Fig 1: Ezh2 inhibition in mESCs causes thermal stability alterations in proteins from the perichromsosmal layer including Mki67, which is methylated in vitro by Ezh2 in K263.A Heatmap showing stability and abundance changes in mESCs and MEFs treated with Ezh2 inhibitors for proteins of the perichromosomal layer55 identified in our AP-MS data as Ezh2 interactors (B). C Pfam domains and prediction of low complexity regions for Mki67. Lys residues in RKS motifs57 as well as Lys residues known to be di- and tri-methylated in vivo58 are shown. The conservation of K263 across different species is shown. D (left) PRM-based quantification of Kme levels (K263me1 and K263me2) in Mki67 in an in vitro methyl-transferase assay using Ezh2. The different peptide sequences used in each quantification are shown. (right) Extracted ion chromatograms (XIC) integrated in Skyline of methylated peptides showing some of the product ions used for quantification. E Comparison of MS/MS spectra between the mono-methylated peptide and its unmodified counterpart. Source data are provided as a Source Data file.
Fig 2: Thermal stability alterations in mitotic chromosome-bound proteins reveal metaphase and chromatin compaction defects upon Ezh2 inhibition.A Scatterplot (left) showing the stability changes measured in mESCs in response to Ezh2 inhibition for 455 proteins known to be stabilized during mitosis in HeLa cells15 (in orange); box-plots (right) showing the distributions of stability (STA) and abundance (ABU) log2 ratios in mESCs and MEFs in response to Ezh2 inhibitors for proteins stabilized in mitotic HeLa cells. B Radial plots showing the median of the log2 ratios for stability (outer circle) and abundance (inner circle) changes in mESCs and MEFs for stabilized (upper) and destabilized (bottom) proteins across cell cycle stages in HeLa cells15. C Heatmap showing the log2 ratios for stability and abundance changes in mESCs and MEFs treated with Ezh2 inhibitors for proteins with known functions in mitotic spindle. D Volcano plots highlighting protein complexes with known functions in mitotic spindle identified as Ezh2 interactors in our AP-MS data. P-values were calculated using limma (two-sided) and adjusted for multiple testing with Benjamini-Hochberg. E Analysis of mitotic phases by immunofluorescence (N = 450 cells). F Localization of Ezh2 in mESCs. This experiment was repeated twice. G Scatterplot (left) showing the stability changes measured in mESCs in response to Ezh2 inhibition for 434 proteins known to be bound to mitotic chromosomes in mESCs54 (in green); box-plots (right) showing the distributions of stability (STA) and abundance (ABU) log2 ratios in mESCs and MEFs in response to Ezh2 inhibitors for proteins bound to mitotic chromosomes. H Examples of metaphases in DMSO- and GSK126-treated mESCs (left). The number of cells with abnormal metaphases is shown on the right (N = 7000 cells; 100 metaphases). I Chromosome area was determined using Ki67, showing greater metaphases upon GSK126 treatment. Ki67 intensity normalized by area showed no differences in Ki67 recruitment to chromosomes during metaphase in response to GSK126. P-values were calculated with a Mann Whitney U test (two-sided). Sample sizes are shown below in parenthesis. For box-plots, median is shown; box limits indicate the 25th and 75th percentiles; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Sample size in each category is shown below in parenthesis. P-values (A, G) are calculated using a non-parametric Wilcoxon test (two-sided). Source data are provided as a Source Data file.
Supplier Page from OriGene Technologies for Ki67 (MKI67) (NM_002417) Human Recombinant Protein