1. Volume 63, Issue 2, Pages 293-305 (July 2016)
Temporal and Spatial Uncoupling of DNA Double Strand Break Repair Pathways within Mammalian Heterochromatin 
Katerina Tsouroula, Audrey Furst, Melanie Rogier, Vincent Heyer, Anne Maglott-Roth, Alexia Ferrand, Bernardo Reina-San-Martin, Evi Soutoglou 
Molecular Cell 
Volume 63, Issue 2, Pages (July 2016)
DOI: /j.molcel
Copyright © 2016 Elsevier Inc. Terms and Conditions

2. Molecular Cell 2016 63, 293-305DOI: (10.1016/j.molcel.2016.06.002)
Copyright © 2016 Elsevier Inc. Terms and Conditions

3. Figure 1
Cas9-Specific Induction of DSBs at Pericentric Heterochromatin
(A) Expression of Cas9-EGFP with a major satellite-specific gRNA in mouse NIH 3T3 cells generates DSBs in pericentric heterochromatin (DAPI-dense regions).
(B) Immunofluorescence (IF) confocal analysis of cells expressing Cas9-EGFP or dCas9-EGFP ± gRNA and stained with DAPI and antibodies specific for γ-H2AX and 53BP1.
(C) IF confocal analysis of cells expressing Cas9-EGFP ± gRNA and stained with DAPI and antibodies specific for 53BP1 and pATMS1981 (top) or γ-H2AX and MDC1 (bottom).
(D) IF confocal analysis of cells expressing Cas9-EGFP+gRNA after treatment with ATM (ATMi) or ATR (ATRi) inhibitor or vehicle (DMSO) and stained with DAPI and a γ-H2AX-specific antibody.
(E) Western blot analysis for Cas9-EGFP (EGFP), γ-H2AX, pATMS1981, pKAP1S824, KAP1, pChk1S345 and tubulin in protein extracts prepared from cells expressing Cas9-EGFP ± gRNA 8 or 16 hr post-transfection. As a comparison, NIH 3T3 cells were treated with increasing concentrations of NCS. Theoretical molecular weights are indicated.
For confocal images, scale bars represent 10 μm. See also Figure S1.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2016 Elsevier Inc. Terms and Conditions

4. Figure 2
Cell-Cycle-Specific Regulation of DSB Localization in Pericentric Heterochromatin
(A) Super-resolution imaging of cells expressing Cas9-EGFP+gRNA and stained with DAPI and antibodies specific for γ-H2AX (top; Movie S1) or 53BP1 (bottom; Movie S2). Quantification is shown on the right.
(B) Super-resolution imaging analysis of G2 (RO-3306-treated) cells expressing Cas9-EGFP+gRNA and stained with DAPI and antibodies specific for γ-H2AX (top; Movie S3) or 53BP1 (bottom; Movie S4). Quantification is shown on the right.
(C) Quantification of γ-H2AX pattern in G1 (EdU−/H3S10p−) and G2 (RO-3306-treated, EdU−/H3S10p+) cells stably expressing Cas9 and transfected with in vitro transcribed major satellite-specific gRNA for the indicated time points and stained with DAPI and antibody specific for γ-H2AX.
For super-resolution images, scale bars represent 5 μm. Values represent mean ± SD of three independent experiments with n = 50 cells. See also Figures S2 and S3.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2016 Elsevier Inc. Terms and Conditions

5. Figure 3
Spatial and Temporal Separation of NHEJ and HR Factor Recruitment in Heterochromatic Structures
(A and B) IF confocal analysis of cells expressing Cas9-EGFP+gRNA specific for major satellite DNA repeats in G1 and G2 and stained with DAPI and antibodies specific for (A) γ-H2AX and RAD51 or (B) γ-H2AX and Ku80. Quantification is shown on the right.
(C and D) IF confocal analysis of cells expressing Cas9-EGFP+gRNA specific for minor satellite DNA repeats in G1 and G2, stained with DAPI and antibodies specific for (C) γ-H2AX and RAD51 or (D) γ-H2AX and Ku80. Quantification of RAD51 or Ku80 recruitment is shown on the right.
Scale bars represent 10 μm. Values represent mean ± SD of three independent experiments with n = 50 cells. See also Figures S4 and S5.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2016 Elsevier Inc. Terms and Conditions

6. Figure 4
Cas9-Induced DSBs Induce Chromatin Expansion Independently of the Release of HP1s or Associated Modifications
(A–I) Schematic representation of the high-throughput analysis (see Supplemental Experimental Procedures) to measure the intensity of γ-H2AX (B), pKAP1S824 (C), HP1α (E), HP1β (F), HP1γ (G), H3K9me3 (H), and KAP1 (I) and the chromocenter (DAPI-dense) area (D). For all plots, individual cell values are represented as box-and-whisker plots (median and quartiles with outliers representing 1% of the population) of two independent experiments with n > 300 cells for G1 and n > 350 cells for G2 (RO-3306 treated cells).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2016 Elsevier Inc. Terms and Conditions

7. Figure 5
Compacted Chromatin Is Not Refractory to the Recruitment of RAD51
(A–C) Quantification of γ-H2AX pattern (A), RAD51 (B), or Ku80 (C) in G1 and G2 cells expressing Cas9-EGFP+gRNA and treated with DMSO or TSA, expressing Cas9-EGFP+gRNA or Cas9-EGFP-VP64+gRNA or depleted for KAP1, HP1α, HP1β, and HP1γ. Values represent mean ± SD of three independent experiments with n = 50 cells. See also Figure S6.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2016 Elsevier Inc. Terms and Conditions

8. Figure 6
DSB Relocation at the Periphery of the Heterochromatin Domain Is Dependent on DNA End Resection
(A) IF confocal analysis of G2 cells expressing Cas9-EGFP+gRNA stained with DAPI and antibodies specific for 53BP1 and RPA32.
(B–E) Quantification of γ-H2AX pattern in G2 cells expressing Cas9-EGFP+gRNA after treatment with DMSO or Mirin inhibitor (B), CtIP knockdown (C), GAM-EmGFP expression (D), or Cas9-CtIP expression (E). (D) IF confocal analysis of G2 cells expressing Cas9-mCherry+gRNA + GAM-EmGFP stained with DAPI and a γ-H2AX-specific antibody is shown.
Scale bars represent 10 μm. Values represent mean ± SD of three independent experiments with n = 50 cells. See also Figures S7A–S7G.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2016 Elsevier Inc. Terms and Conditions

9. Figure 7
The RAD51/BRCA2 Complex Stabilizes DSBs at the Periphery of Heterochromatin
(A and B) Quantification of RAD51 recruitment (A) and γ-H2AX pattern (B) in G2 cells expressing Cas9-EGFP+gRNA under BRCA2 knockdown conditions.
(C) Quantification of γ-H2AX pattern in G2 cells expressing Cas9-RAD51 (left) or Cas9-BRC3 or Cas9-mBRC3 (right).
(D) IF confocal analysis of G2 cells expressing Cas9-RAD51 or Cas9-BRC3+gRNA stained with DAPI and specific antibodies for γ-H2AX and RAD51.
(E) IF confocal analysis of G1 and G2 cells expressing Cas9-mCherry + gRNA + RAD52-EGFP stained with DAPI and specific antibodies for γ-H2AX and RAD51.
(F) Quantification of RAD52 recruitment and pattern in G1 and G2 expressing Cas9-mCherry + gRNA + RAD52-EGFP.
(G) Quantification of γ-H2AX pattern in G2 cells expressing Cas9-EGFP + gRNA under RAD52 knockdown conditions.
Scale bars represent 10 μm. Values represent mean ± SD of three independent experiments with n = 50 cells. See also Figures S7H–S7M.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2016 Elsevier Inc. Terms and Conditions