Journal: Molecular cell

Article Title: The BUB3-BUB1 Complex Promotes Telomere DNA Replication

doi: 10.1016/j.molcel.2018.03.032

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse anti-BUB3 BD Bioscience Cat# 611730 Mouse anti-BUB1 Abcam Cat# ab54893 Rabbit anti-BUBR1 BD Bioscience Cat# 612502 Mouse anti-HA Abcam Cat# ab18181 Mouse monoclonal anti-FLAG (M2) Sigma-Aldrich Cat# F3165 Mouse monoclonal anti-GST Abmart Cat# {"type":"entrez-nucleotide","attrs":{"text":"M20007","term_id":"172509"}} M20007 Rabbit polyclonal anti-FLAG Sigma-Aldrich Cat# F7425 Rabbit anti-BLM Abcam Cat# ab2179 Rabbit anti-GFP Abcam Cat# ab6556 Rabbit anti-POT1 Songyang laboratory N/A Rabbit anti-PCNA Abcam Cat# ab19166 Mouse monoclonal anti-TIN2 Songyang laboratory N/A Rabbit anti-GAPDH ABclonal Cat# AC027 Rabbit polyclonal anti-RAP1 Songyang laboratory N/A Bacterial and Virus Strains BL21(DE3) NEW ENGLAND BioLabs Cat# C2527I TOP10 Competent E. coli Invitrogen Cat# C404003 Sf9 cells Thermo Fisher Scientific Cat# 11496015 Chemicals, Peptides, and Recombinant Proteins 2OH-BNPP1 Kang et al., 2008 N/A Nocodazole Sigma Cat# M1404 Proteinase inhibitor Cocktail Sigma Cat# P8340 TelC-Alexa488 PNA Bio Cat# F1004 TelG-Cy3 PNA Bio Cat# F1006 LR ClonaseTM II Plus enzyme Thermo Fisher Scientific Cat# 12538120 Critical Commercial Assays BCA Protein Assay Kit Pierce Cat# 23225 Click-iTTM EdU Alexa FluorTM 488 Imaging Kit Thermo Fisher Scientific Cat# {"type":"entrez-nucleotide","attrs":{"text":"C10337","term_id":"1535408"}} C10337 Lipofectamine® RNAiMAX NEW ENGLAND BioLabs Cat# 13778150 LipofectamineTM 3000 Thermo Fisher Scientific Cat# L3000015 SYBRGreen PCR Master Mix ABI Cat# 4368708 Deposited Data Mass Spectrometry This paper; and Table S1 Experimental Models: Cell Lines HeLa229 ATCC CCL-2.1 293T ATCC CLR3216 HTC75 Yang. et al., 2016 Oligonucleotides siRNA targeting sequences, see Table S2 This paper N/A gRNA targeting sequences, see Table S2 This paper N/A Primer sequences, see Table S2 This paper N/A Recombinant DNA Plasmid: pDEST15 Invitrogen Cat# 11802014 Plasmid: pDEST17 Invitrogen Cat# 11803012 Plasmid: pDEST27 Invitrogen Cat# 11812013 Plasmid: PX330 Addgene Cat# 42230 Plasmid: pENTRTM/D-TOPOTM Invitrogen Cat# K240020 Plasmid: pET28-His-TEVs This paper N/A Plasmid: pBabe-CMV-DEST-SFB This paper N/A Plasmid: pBabe-CMV-DEST-GFP This paper N/A Plasmid: pBabe-CMV-DEST-HA-Flag This paper N/A Plasmid: PITCh-SFB-GFP This paper N/A Software and Algorithms ImageJ, Fiji Available online https://fiji.sc/ NIS-Elements Viewer Available online https://en.freedownloadmanager.org/Windows-PC/NIS-Elements-Viewer-FREE.html ZEISS- AxioVision Available online https://www.zeiss.com/microscopy/int/products/microscope-software/axiovision.html Open in a separate window KEY RESOURCES TABLE

Techniques: Virus, Recombinant, Bicinchoninic Acid Protein Assay, Imaging, Mass Spectrometry, Plasmid Preparation, Software

Journal: Scientific Reports

Article Title: LIM Protein Ajuba associates with the RPA complex through direct cell cycle-dependent interaction with the RPA70 subunit

doi: 10.1038/s41598-018-27919-8

Figure Lengend Snippet: Ajuba is in association with the RPA complex, and dissociates upon hydroxyurea treatment. ( A ) IP-Western on immunoprecipitations with Ajuba serum from HTC75 total extracts. The blots were probed with the indicated RPA antibodies. ( B ) IP-Western using the indicated RPA antibodies for immunoprecitations, and blotted for Ajuba. ( C ) IP-Western using anti-Ajuba antibodies for immunoprecipitations, and blotted for RPA32. Total extracts shown on the left panel. Cells were untreated (control), treated with 2 mM hydroxyurea (HU) for 24 hr, or synchronized by double Thymidine block and released for 2 hr. See also Figures , and for data with IMR90 cells. Panels shown are cropped from full-length blots shown in Supplemental Figure .

Article Snippet: HTC75 cells were cultured in DMEM (Cellgro) with 10% BCS (HyClone), 1% penicillin and streptomycin (Cellgro) and 1% L-glutamine (Gibco).

Techniques: Western Blot, Control, Blocking Assay

Journal: Scientific Reports

Article Title: LIM Protein Ajuba associates with the RPA complex through direct cell cycle-dependent interaction with the RPA70 subunit

doi: 10.1038/s41598-018-27919-8

Figure Lengend Snippet: Ajuba nuclear localization is increased during S phase. HTC75 cells were synchronized to G1/S border with double thymidine block and released into S phase, with cells processed for IF at the indicated time points. (Top) Immunofluorescence of Ajuba in unsynchronized and synchronized cells. (Bottom) Quantitation of cells positive for nuclear Ajuba at each time point (n = 100).

Article Snippet: HTC75 cells were cultured in DMEM (Cellgro) with 10% BCS (HyClone), 1% penicillin and streptomycin (Cellgro) and 1% L-glutamine (Gibco).

Techniques: Blocking Assay, Immunofluorescence, Quantitation Assay

Journal: Scientific Reports

Article Title: LIM Protein Ajuba associates with the RPA complex through direct cell cycle-dependent interaction with the RPA70 subunit

doi: 10.1038/s41598-018-27919-8

Figure Lengend Snippet: Nuclear Ajuba is found primarily in BrdU-positive cells. HTC75 cells were synchronized, then released in BrdU-containing medium. The BrdU was washed off after 1 hour. Cells were co-stained for Ajuba and BrdU at the indicated time points. The percent of nuclei with >3 foci of co-localization is shown (bottom right).

Article Snippet: HTC75 cells were cultured in DMEM (Cellgro) with 10% BCS (HyClone), 1% penicillin and streptomycin (Cellgro) and 1% L-glutamine (Gibco).

Techniques: Staining

Journal: Scientific Reports

Article Title: LIM Protein Ajuba associates with the RPA complex through direct cell cycle-dependent interaction with the RPA70 subunit

doi: 10.1038/s41598-018-27919-8

Figure Lengend Snippet: Increase of Ajuba-RPA70 co-localization in the nucleus during S phase. HTC75 cells were synchronized to G1/S border with double thymidine block and released into S phase with cells processed for IF at the indicated time points. (Top) Co-immunofluorescence of Ajuba and RPA70 in unsynchronized and synchronized cells. Arrowheads point to sites of co-localization (Bottom) Quantitation of cells exhibiting >3 foci of Ajuba-RPA70 co-localization in the nucleus at each time point (n = 100).

Article Snippet: HTC75 cells were cultured in DMEM (Cellgro) with 10% BCS (HyClone), 1% penicillin and streptomycin (Cellgro) and 1% L-glutamine (Gibco).

Techniques: Blocking Assay, Immunofluorescence, Quantitation Assay

Journal: Scientific Reports

Article Title: LIM Protein Ajuba associates with the RPA complex through direct cell cycle-dependent interaction with the RPA70 subunit

doi: 10.1038/s41598-018-27919-8

Figure Lengend Snippet: Ajuba nuclear localization is reduced upon HU treatment. (Left) Co-immunofluorescence of Ajuba (FITC) and RPA70 (TRITC) in untreated and HU treated HTC75 cells. (Right) Quantification of cells exhibiting strong nuclear localization with and without HU treatment (n = 100, three independent experiments).

Article Snippet: HTC75 cells were cultured in DMEM (Cellgro) with 10% BCS (HyClone), 1% penicillin and streptomycin (Cellgro) and 1% L-glutamine (Gibco).

Techniques: Immunofluorescence

Journal: Cell reports

Article Title: Shwachman-Diamond Syndrome Protein SBDS Maintains Human Telomeres by Regulating Telomerase Recruitment

doi: 10.1016/j.celrep.2018.01.057

Figure Lengend Snippet: (A) The inducible SBDS-knockout HeLa cells were harvested 6 days after doxycycline treatment (1 μg/mL) for western blot analysis. Endogenous SBDS protein levels were quantified using the Odyssey Infrared Imaging System and normalized against the GAPDH loading control.

Article Snippet: HEK293T, HTC75, and HeLa cells were cultured in DMEM (Corning Life Sciences) supplemented with 10% fetal bovine serum (Gibco) and 100 U/mL streptomycin/penicillin (Gibco) at 37°C in a 5% CO 2 incubator.

Techniques: Knock-Out, Western Blot, Imaging, Control

Journal: Molecular Biology of the Cell

Article Title: Persistent telomere cohesion triggers a prolonged anaphase

doi: 10.1091/mbc.E13-08-0479

Figure Lengend Snippet: Tankyrase 1 depletion by siRNA leads to anaphase delay in HTC75 cells. ( A–D) Centromeres separate in tankyrase 1–depleted cells. (A) Schematic diagram depicting persistent telomere cohesion and loss of centromere cohesion induced by TNKS1 depletion. (B–D) HTC75 cells treated with GFP or TNKS1 siRNA for 48 h were analyzed by (B) immunoblot and (C) centromere FISH (red) after mitotic shake-off. (D) Graphical representation of the frequency of mitotic cells with centromeres apart. Values are means ± SEM derived from two independent experiments ( n = 30–100 cells each). (E–G) Wild-type (WT) but not PARP-dead tankyrase 1 rescues centromere separation. Stable HTC75 cell lines expressing GFP or TNKS1 shRNA were transfected with a vector control or siRNA resistant (r) TNKS1 WT or PARP-dead plasmids and analyzed by (E) immunoblot and (F) centromere (red) and telomere (green) FISH after mitotic shake-off. (G) Graphical representation of the frequency of mitotic cells with telomeres cohered and centromeres apart. Values are means ± SEM, derived from two independent experiments ( n = 50–146 cells each). (C, F) DNA was stained with DAPI (blue). Scale bars, 5 μm. (H–L) Time-lapse video live-cell imaging of a HTC75-H2B-GFP cell line 36 h after transfection with GFP or TNKS1 siRNA. (H) Progression from prophase to anaphase for individual cells. Scale bar, 5 μm. (I–L) Graphical summaries of individual mitotic cells ( n = 20–21 cells from two independent experiments) shown as (I) a time line and (J–L) scatterplots with calculated mean value ± SEM. Student's t test was used to calculate p values (ns, p ≥ 0.05; ** p ≤ 0.01; *** p ≤ 0.001).

Article Snippet: HTC75 cells (an HT1080-derived clonal cell line; ), IMR90 PD 20 (ATCC), TIN2C (an HTC75 cell line overexpressing TIN2C; Canudas et al ., 2011 ), and SA1 (an HTC75 cell line overexpressing SA1; Bisht et al ., 2013 ) were infected and selected in 2 μg/ml puromycin as described ( Houghtaling et al ., 2004 ).

Techniques: Western Blot, Derivative Assay, Expressing, shRNA, Transfection, Plasmid Preparation, Control, Staining, Live Cell Imaging

Journal: Molecular Biology of the Cell

Article Title: Persistent telomere cohesion triggers a prolonged anaphase

doi: 10.1091/mbc.E13-08-0479

Figure Lengend Snippet: TIN2C or SA1 overexpression leads to anaphase delay. (A) Schematic diagram depicting persistent telomere cohesion and loss of centromere cohesion induced by TIN2C or SA1 overexpression. (B, C, G, H) TIN2C and SA1 overexpression leads to a loss in centromere cohesion. HTC75 cells stably expressing (B, C) V or TIN2C or (G, H) V or SA1 were analyzed by (B, G) centromere (red) and telomere (green) FISH after mitotic shake-off. DNA was stained with DAPI (blue). Scale bars, 5 μm. (C, H) Graphical representation of the frequency of mitotic cells with centromeres apart and telomeres cohered. Values are means ± SEM, derived from two independent experiments (C, n = 50–56 cells each; H, n = 50–60 cells each). (D–F, I–K) Live-cell imaging indicates that TIN2C or SA1 overexpression induces anaphase delay. Time-lapse video live-cell imaging of (D–F) HTC75.Vector-H2B-GFP and HTC75.TIN2C-H2B-GFP or (I–K) HTC75.Vector-H2B-GFP and HTC75.SA1-H2B-GFP cell lines. (D, I) Progression from prophase to anaphase for individual cells. Scale bars, 5 μm. (E, F, J, K) Graphical summaries of individual mitotic cells (E, F; n = 49–56 cells each from two independent experiments) and (I–K; n = 18–21 cells each) shown as (E, J) a time line and (F, K) a scatterplot with calculated mean value ± SEM. Student's t test was used to calculate p values (*** p ≤ 0.001; **** p ≤ 0.0001).

Article Snippet: HTC75 cells (an HT1080-derived clonal cell line; ), IMR90 PD 20 (ATCC), TIN2C (an HTC75 cell line overexpressing TIN2C; Canudas et al ., 2011 ), and SA1 (an HTC75 cell line overexpressing SA1; Bisht et al ., 2013 ) were infected and selected in 2 μg/ml puromycin as described ( Houghtaling et al ., 2004 ).

Techniques: Over Expression, Stable Transfection, Expressing, Staining, Derivative Assay, Live Cell Imaging, Plasmid Preparation

Journal: Molecular Biology of the Cell

Article Title: Persistent telomere cohesion triggers a prolonged anaphase

doi: 10.1091/mbc.E13-08-0479

Figure Lengend Snippet: Tankyrase 1 overexpression rescues persistent telomere cohesion, regardless of the mechanism of induction. (A–H) Tankyrase 1 overexpression rescues persistent telomere cohesion and loss of centromere cohesion in TIN2C- or SA1-overexpressing cells. (A, E) Schematic diagrams. (B–D) HTC75.V or TIN2C and (F–H) HTC75.V or SA1 cells were transfected for 24 h with a vector or TNKS1 plasmid and analyzed by (B, F) immunoblot and (C, G) centromere (red) and telomere (green) FISH after mitotic shake-off. DNA was stained with DAPI (blue). Scale bars, 5 μm. (D, H) Graphical representation of the frequency of mitotic cells with centromeres apart and telomeres cohered (D, n = 50–56 cells each; H, n = 50–60 cells each). (I–L) Wild-type but not PARP-dead tankyrase 1 rescues telomere cohesion and centromere separation in aging IMR90 cells. (I) Schematic diagram. (J–L) IMR90 cells stably expressing tankyrase 1 WT or PARP dead or a vector control were generated by lentiviral infection at an early PD (20). Cell lines were grown for multiple PDs and then, before senescence (PD 48), analyzed by (J) immunoblot and (K) centromere (red) and telomere (green) FISH after mitotic shake-off. DNA was stained with DAPI (blue). Scale bar, 5 μm. (L) Graphical representation of the frequency of mitotic cells with telomeres cohered and centromeres apart. Values are means ± SEM, derived from two independent experiments ( n = 50 cells each). (M) Loss of synchrony in sister telomere separation in aging IMR90 cells can be rescued by wild-type but not PARP-dead tankyrase 1. Graphical representation of the frequency of mitotic cells with one singlet/one doublet, two singlets, or two doublets in IMR90 cells expressing vector, TNKS1.WT, or TNKS1.PARP dead at PD 48. Values are means ± SEM, derived from two independent experiments ( n = 50 cells each). (N) Model showing that excess telomere cohesion leads to anaphase delay.

Article Snippet: HTC75 cells (an HT1080-derived clonal cell line; ), IMR90 PD 20 (ATCC), TIN2C (an HTC75 cell line overexpressing TIN2C; Canudas et al ., 2011 ), and SA1 (an HTC75 cell line overexpressing SA1; Bisht et al ., 2013 ) were infected and selected in 2 μg/ml puromycin as described ( Houghtaling et al ., 2004 ).

Techniques: Over Expression, Transfection, Plasmid Preparation, Western Blot, Staining, Stable Transfection, Expressing, Control, Generated, Infection, Derivative Assay