1. Volume 2, Issue 6, Pages 1521-1529 (December 2012)
Human Telomeres Are Tethered to the Nuclear Envelope during Postmitotic Nuclear Assembly 
Laure Crabbe, Anthony J. Cesare, James M. Kasuboski, James A.J. Fitzpatrick, Jan Karlseder 
Cell Reports 
Volume 2, Issue 6, Pages (December 2012)
DOI: /j.celrep
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2. Cell Reports 2012 2, 1521-1529DOI: (10.1016/j.celrep.2012.11.019)
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3. Figure 1 Live Cell Imaging of Human Telomeres
(A) Time-lapse images of HeLa cells expressing EGFP-TRF1 and H2B-mCherry. A single focal plane is presented per time-lapse (hr:min).
(B) Magnification of the 6:03 and 7:03 images.
(C) Immunofluorescence staining of synchronized HeLa cells expressing EGFP-TRF1 in G1/S, S phase, or after mitosis (Next G1).
(D) Magnification of two nuclei from (C), Next G1.
(E) Staining of synchronized IMR90 cells expressing EGFP-TRF1 after mitosis (Next G1).
(F) FISH staining of HeLa cells.
See also Figures S1 and S2, and Movies S1 and S2.
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4. Figure 2
Quantification of Telomere and Centromere Position in the Nucleus
(A) Schematic of the quantification method. The ratio between x (distance from the signal of interest to the center) and R (radius of the ellipse through that signal) is calculated. This ratio allows to position the signal in one of the three zones of equal surface I, II, and III.
(B) Representative example of a 2D reconstruction of telomeres and centromeres and their classification into three zones. Telomeres and centromeres that belong to each specific zone are displayed in yellow. The center of the nucleus is displayed in red.
(C) Quantification of telomere and centromere position in the nucleus of HeLa cells. G1, S phase, and Next G1 data are represented as a percentage of spots (y axis) per zone (x axis); as mean ± SD. The number of nuclei, telomeres, and centromeres is shown in Table S1.
(D) Immunofluorescence staining of synchronized HeLa cells stably expressing EGFP-TRF1 (maximum intensity projection).
(E) Magnification of two individual nuclei from (D) (Next G1).
See also Figure S2.
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5. Figure 3 Telomeres Are Physically Tethered to the Nuclear Envelope
(A) Telomere association assay of HeLa cells expressing NLS-EGFP or EGFP-TRF1. Cells were collected at the G1/S boundary or 10 and 12 hr after release.
(B) Quantification of signals from (A).
(C) Coimmunoprecipitation of HeLa cells expressing, Myc alone, Myc-RAP1, and/or EGFP-Sun1. Myc beads or GFP beads were used for immunoprecipitation.
(D) Coimmunoprecipitation of HeLa cells expressing Myc-TRF2 or Flag-TRF1 and EGFP-Sun1. Myc beads or GFP beads were used for immunoprecipitation as indicated (IP MYC, IP EGFP).
See also Figure S3.
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6. Figure 4 RAP1-Independent Telomere Tethering
(A) Western blotting of HeLa cells transfected with Sun1 or a control siRNA.
(B) Time-lapse images from movies of HeLa cells stably expressing EGFP-TRF1 and H2B-mCherry and transfected with Sun1 siRNA. A single focal plane is presented per time-lapse (indicated in hr:min).
(C) Schematic of the Sun1-NΔ200 mutant and immunofluorescence of cells expressing EGFP-TRF1 and Sun1 wt or Sun1-NΔ200-mCherry.
(D) Time-lapse images from movies of HeLa cells stably expressing EGFP-TRF1 and transfected with Sun1-NΔ200-mCherry. A maximum intensity projection of all focal planes is presented per time-lapse (hr:min).
(E) Western blotting of HeLa cells expressing RAP1 or control shRNA.
(F) Quantification of telomere position in the nucleus after mitosis of HeLa cells. Nuclei after mitosis were selected from time-lapse images and used for quantification. Data are represented as a percentage of spots (y axis) per zone (x axis); as mean ± SD. The number of nuclei and telomeres is shown in Table S1.
See also Movie S3.
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7. Figure S1
Expression of Tagged Telomeric Proteins in HeLa and IMR90 Cells, Related to Figure 1
(A) Immunofluorescence staining of HeLa cells expressing EGFP-TRF1 (green). TRF2 has been labeled in red and the DNA in blue.
(B) Time-lapse images from movies of HeLa or IMR90 cells stably expressing EGFP-TRF1 (green) and H2B-mCherry (red). A single focal plane is shown.
(C) Western Blotting of IMR90 or HeLa cells expressing EGFP-TRF1 or H2B-mCherry as indicated. HeLa were irradiated with 2.5 Gy γ-irradiation when indicated. Antibodies used are indicated on the left. The arrow points to the band for EGFP-TRF1. The arrowhead points to the band for endogenous TRF1.
(D) Time-lapse images from movies of IMR90 cells stably expressing EGFP-TRF1 (green) and H2B-mCherry (red). A single focal plane is presented per time-lapse (indicated in hr:min). Cell cycle phases have been indicated on the left.
(E) FACS profiles of synchronized HeLa cells stably expressing NLS-EGFP or EGFP-TRF1.
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8. Figure S2
Telomere Localization in Primary Cells, Related to Figures 1 and 2
(A) Representative example of a 2D reconstruction of telomeres from IMR90 (green/yellow dots) of two nuclei after mitosis (blue), and their classification into one of the three zones as indicated. Telomeres that belong to each specific zone are displayed in yellow. The center of the nuclei are displayed in red.
(B) Immunofluorescence staining of IMR90 cells expressing EGFP-TRF1 (green). Lamin A/C has been labeled in red and the DNA in blue. The arrows point to invagination of the nuclear envelope membrane into the nucleus.
(C) Quantification of telomere position in the nucleus of IMR90 cells derived from immunofluorescence confocal images. For each stage of the cell cycle (G1, S phase and Next G1), data are represented as a percentage of spots (y axis) per zone (x axis); as mean. The number of nuclei analyzed is indicated for each cell cycle phase.
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9. Figure S3
Biochemical Interactions of Telomeric Proteins with Factors of the Nuclear Envelope, Related to Figure 3
(A) ChIP experiments from synchronized HeLa in G1-S, S phase, or 12 hr after release (Next G1). Antibodies used are indicated on the left, and 10% of the input is shown. DNA was subjected to Southern blot analysis using telomeric (left panel) or ALU repetitive probes (right panel).
(B) Immunofluorescence staining of HeLa cells in G1/S or after mitosis (Next G1) expressing EGFP-TRF1 (green). Sun1 has been labeled in red. Arrows point to aggregates of Sun1 stored in the endoplasmic reticulum.
(C) Telomere association assay of HeLa cells stably expressing NLS-EGFP (panel IP NLS) or EGFP-TRF1 (panel IP TRF1). GFP beads were used for immunoprecipitation from lysates of cells collected at the G1/S block, in Sphase, or 12 hr after release. Antibodies used are indicated on the left.
(D) Quantification of the telomere signal from panel B.
(E) Telomere association assay of HeLa cells stably expressing EGFP-TRF1. MYC beads were used for immunoprecipitation in lysates from cells collected at the G1/S block, in Sphase, or 12 hr after release. Antibodies used are indicated on the left.
(F) Co-immunoprecipitation of HeLa cells not transfected (NT) or expressing EGFP-Sun1. GFP beads were used for immunoprecipitation. Antibodies used are indicated on the left.
(G) Co-immunoprecipitation of HeLa cells expressing Myc-TRF2 and EGFP-Sun1. Myc beads or GFP beads were used for immunoprecipitation as indicated (IP MYC, IP EGFP). Antibodies used are indicated on the left.
Cell Reports 2012 2, DOI: ( /j.celrep )
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