Extended Data Fig. 6: Characterization of USP11 role in licensing of HR at centromeric DSBs in G1.
From: Activation of homologous recombination in G1 preserves centromeric integrity
a, Western blot analysis of Ubiquitin (FK2 antibody) after GFP-IP under denaturing conditions in NIH3T3 cells expressing GFP or GFP-CENPA and co-expressing USP11-Flag vs Flag. The input was 1% of the extract used for the IP. b, Western blot analysis of ubiquitin (FK2 antibody) after GFP-IP under denaturing conditions in NIH3T3 cells expressing GFP or GFP-HJURP and siUSP11 vs siSCR. The input was 1% of the extract used for the IP. c, 2 biological replicates of western blot analysis showing the interaction of CENPA with HJURP in siUSP11 vs siSCR NIH3T3 cells (left panel). Quantification from western blot of CENPA interaction with HJURP in siUSP11 vs siSCR NIH3T3 cells (right panel). d, Quantification of fold change of CENP-A recruitment at LacO in cells expressing LacI-HJURP and co-expressing siUSP11 vs siSCR. e, Schematic representation of the SNAP technology. f, Western blot analysis of USP11, GFP and tubulin in HeLa cells expressing GFP-HJURP or not and co-expressing siUSP11 or siHJURP vs siSCR. g, Immunofluorescence confocal analysis of de novo deposited CENP-A-SNAP (TMR Star in Red) and total CENPA (Green) in Hela expressing CENP-A-SNAP and co-expressing siHJURP or siUSP11 vs siSCR. h, Cell cycle analysis by flow cytometry using propidium iodide and EdU in HeLa CENP-A-SNAP cells depleted of USP11 relative to siSCR control. i, Chromatin immunoprecipitation analysis of USP11 enrichment at pericentromeric heterochromatin over the Input and relative to the no-antibody control, in NIH3T3 cells expressing Cas9 + mi gRNA vs dCas9 + gRNA (left panel). Enrichment of USP11 at GAPDH promoter over the Input and relative to the no-antibody control, in NIH3T3 cells expressing Cas9 + gRNA targeting GAPDH promoter vs dCas9 + gRNA (right panel). j, Chromatin immunoprecipitation analysis of CENPA (left) and Histone H3 (right) enrichment at centromeres over the Input and relative to the no-antibody control, in NIH3T3 cells expressing Cas9 + mi gRNA vs dCas9 + mi gRNA. k, IF confocal analysis of de novo deposited CENP-A-SNAP in Hela expressing CENP-A-SNAP and GFP-dCas9 (left) or Cas9 and stained with γ-H2AX antibody (right). l, Quantification of fold change of RAD51 recruitment in G1 at pericentromeric DSBs in NIH3T3 cells expressing Cas9 + gRNA targeting major satellite repeats, co-expressing dCas9-CENPA (upper panel) or dCas9-HJURP (lower panel) and depleted of USP11 (siUSP11) vs siSCR. For confocal images, scale bars represent 5 µm. For definition of p values, statistic method and sample number see Statistics and Reproducibility section.
