Journal:

Article Title: Telomerase-associated Protein 1, HSP90, and Topoisomerase II? Associate Directly with the BLM Helicase in Immortalized Cells Using ALT and Modulate Its Helicase Activity Using Telomeric DNA Substrates * S⃞

doi: 10.1074/jbc.M900195200

Figure Lengend Snippet: Identification of BLM- and TRF2-associated proteins by mass spectrometry. BLM-associated proteins were immunoprecipitated from immortalized telomerase-positive (HeLa and MCF7, lanes 3 and 5) and telomerase-negative ALT cells (WI38-VA13 and Saos2, lanes 4 and 6) using anti-BLM antibody. Lysates were subjected to a second round of immunoprecipitation using anti-TRF2 to enrich for BLM-associated proteins in cells that use ALT. Lanes 1 and 2 are IgG controls for MCF7 and Saos2 cells. Immunoprecipitates were resolved by 10% SDS-PAGE and silver-stained. Proteins identified by mass spectrometry are labeled.

Article Snippet: Antibodies were purchased from Accurate Scientific (BrdUrd, OBT 0030), Bethyl Laboratories (BLM, A300-110A for immunoprecipitations and Western blots; TOPOIIα, A300-054A for immunoprecipitations and Western blots), Calbiochem (TOPOIIα, NA14 for immunofluorescence), Imgenex (TRF2, IMG-124A for immunofluorescence), and Santa Cruz Biotechnology (HSP90, sc-1057; PML, PG-M3; TEP1, sc-13052).

Techniques: Mass Spectrometry, Immunoprecipitation, SDS Page, Staining, Labeling

Journal:

Article Title: Telomerase-associated Protein 1, HSP90, and Topoisomerase II? Associate Directly with the BLM Helicase in Immortalized Cells Using ALT and Modulate Its Helicase Activity Using Telomeric DNA Substrates * S⃞

doi: 10.1074/jbc.M900195200

Figure Lengend Snippet: Detection of BLM and TRF2-associated proteins by Western analysis. A, BLM and TRF2 were coimmunoprecipitated sequentially from nuclear extracts of ALT (WI38-VA13 and Saos2) and telomerase-positive (HeLa and MCF7) or BS cells (GM08508) (panels a–c). Immunoprecipitations (IP) were carried out with goat polyclonal BLM and TRF2 antibodies or normal goat IgG (negative control). Immunoprecipitates were resolved by 6% SDS-PAGE, Western-blotted with anti-TEP1 (a), anti-TOPOIIα (b), and anti-HSP90 (c) antibodies. TEP1 (d), TOPOIIα (e), and HSP90 (f) were also immunoprecipitated from nuclear extracts of Saos2 and MCF7 cells, resolved, and probed with anti-BLM and anti-TRF2. IgG controls and the inputs are also shown. B, TRF1 associates with BLM and TRF2. BLM·TRF2 immunocomplex from Saos2 and MCF7 were subjected to Western analysis using anti-TRF1 antibody. TRF1 immunoprecipitated from the ALT cell line Saos2. BLM was detected in the immunocomplex from both cell lines, whereas TRF2 was present in complex with BLM and TRF1 in Saos2. C, TEP1 is not a component of the ALT complex containing TRF1. Whole cell lysates from Saos2 and MCF7 were subjected to sequential immunoprecipitation using anti-BLM and anti-TRF1 antibodies. HSP90, TOPOIIα, and TRF2 were detected in complex with BLM and TRF2 in Saos2 but not in MCF7. TEP1 was not included in this specific complex in either cell line. The Input lanes show the presence of the proteins in whole cell lysates.

Article Snippet: Antibodies were purchased from Accurate Scientific (BrdUrd, OBT 0030), Bethyl Laboratories (BLM, A300-110A for immunoprecipitations and Western blots; TOPOIIα, A300-054A for immunoprecipitations and Western blots), Calbiochem (TOPOIIα, NA14 for immunofluorescence), Imgenex (TRF2, IMG-124A for immunofluorescence), and Santa Cruz Biotechnology (HSP90, sc-1057; PML, PG-M3; TEP1, sc-13052).

Techniques: Western Blot, Negative Control, SDS Page, Immunoprecipitation

Journal:

Article Title: Telomerase-associated Protein 1, HSP90, and Topoisomerase II? Associate Directly with the BLM Helicase in Immortalized Cells Using ALT and Modulate Its Helicase Activity Using Telomeric DNA Substrates * S⃞

doi: 10.1074/jbc.M900195200

Figure Lengend Snippet: TEP1, TOPOIIα, and HSP90 co-localize with BLM and TRF2 in ALT cells in vivo. Three proteins co-localize with BLM (A) and TRF2 (B) in immortalized ALT cells Saos2. A, for generating immunofluorescence with BLM, cells were transfected with pEGFP-BLM, fixed, and immunostained using rabbit polyclonal anti-TEP1, anti-TOPOIIα, and anti-HSP90, and rhodamine-labeled secondary. Negative controls with rabbit IgG are shown. DAPI, 4′,6-diamidino-2-phenylindole. B, immunofluorescence with TRF2- and BLM-associated ATL complex proteins were performed as described under “Experimental Procedures.” Immunostaining was performed using mouse monoclonal anti-TRF2 and rabbit polyclonal antibodies against the BLM-associated proteins. Mouse IgG was used for negative controls. BLM (A) and TRF2 (B) merge with TEP1 or TOPOIIα or HSP90 to give yellow foci in ALT cell line Saos2. Strong co-localization was not observed in the telomerase-positive cell line MCF7. C, quantitation of A and B. % of cells showing co-localization of BLM or TRF2 with the BLM-associated ALT complex proteins were calculated from three independent experiments to obtain averages and S.D.

Article Snippet: Antibodies were purchased from Accurate Scientific (BrdUrd, OBT 0030), Bethyl Laboratories (BLM, A300-110A for immunoprecipitations and Western blots; TOPOIIα, A300-054A for immunoprecipitations and Western blots), Calbiochem (TOPOIIα, NA14 for immunofluorescence), Imgenex (TRF2, IMG-124A for immunofluorescence), and Santa Cruz Biotechnology (HSP90, sc-1057; PML, PG-M3; TEP1, sc-13052).

Techniques: In Vivo, Immunofluorescence, Transfection, Labeling, Immunostaining, Quantitation Assay

Journal:

Article Title: Telomerase-associated Protein 1, HSP90, and Topoisomerase II? Associate Directly with the BLM Helicase in Immortalized Cells Using ALT and Modulate Its Helicase Activity Using Telomeric DNA Substrates * S⃞

doi: 10.1074/jbc.M900195200

Figure Lengend Snippet: BLM knockdown and TRF length analysis in cells using ALT (Saos2) or telomerase (MCF7) to maintain telomeres demonstrate the requirement for BLM in ALT. A, Saos2 and MCF7 cells were transfected with pBLMsiRNA and control pSCsiRNA (negative or untreated control) and cultured for 3 weeks in the presence of the selective antibiotic puromycin (6 ng/μl). Stably transfected clones were harvested and analyzed by Western analysis using rabbit anti-BLM (Bethyl Laboratories). B, genomic DNAs were isolated and analyzed by Southern blot. TRF lengths are decreased in average size in Saos2 cells after siRNA treatment against BLM, in comparison to the treated controls. This decrease was not observed in MCF7 cells treated similarly. Size markers are shown to the left. Kbp, kilobase pairs. C, BLM knockdown affects the co-localization of the BLM-associated proteins with APBs and TRF2 in ALT cells. Co-localization of TEP1, TOPOIIα, and HSP90 with APBs or TRF2 was monitored by immunofluorescence in the ALT cell line Saos2 transiently transfected with control or pBLMsiRNA as described under “Experimental Procedures.” Percent co-localization was determined as before (Fig. 3C). D, BLM knockdown does not affect the expression level of TEP1, HSP90, and TOPOIIα. Total cell lysates prepared from control or pBLMsiRNA-transfected Saos2 and MCF7 cell lines were resolved by 10% SDS-PAGE and Western-analyzed using anti-TEP1, anti-HSP90, and anti-TOPOIIα. α-Actin was used for the loading control.

Article Snippet: Antibodies were purchased from Accurate Scientific (BrdUrd, OBT 0030), Bethyl Laboratories (BLM, A300-110A for immunoprecipitations and Western blots; TOPOIIα, A300-054A for immunoprecipitations and Western blots), Calbiochem (TOPOIIα, NA14 for immunofluorescence), Imgenex (TRF2, IMG-124A for immunofluorescence), and Santa Cruz Biotechnology (HSP90, sc-1057; PML, PG-M3; TEP1, sc-13052).

Techniques: Transfection, Cell Culture, Stable Transfection, Clone Assay, Western Blot, Isolation, Southern Blot, Comparison, Immunofluorescence, Expressing, SDS Page

Journal: Frontiers in Cell and Developmental Biology

Article Title: Loss of FAM111B protease mutated in hereditary fibrosing poikiloderma negatively regulates telomere length

doi: 10.3389/fcell.2023.1175069

Figure Lengend Snippet: FAM111B -deficient cells have shorter telomeres. (A) Widefield images of chromosome spreads and (B) interphase cells from wild-type (WT) and FAM111B knockout cells stained with fluorescent probe against telomeric repeats. Scale bar 20 μm. (C) Quantification of individual TeloFISH foci intensity in metaphase spreads and interphase cells. Green bars show averages of N = 3, with at least 30 metaphase spreads or 60 cells scored per sample in each experiment. (D) Widefield images of wild-type (WT) and FAM111B −/- U2OS cells stained with TRF2 antibodies. Scale bar 20 μm. (E) Quantification of TRF2 foci intensity in U2OS wild-type (WT) FAM111B negative U2OS cells. Green bars show averages of N = 3 experiments, where 400 cells was scored per sample in each experiment. (F) Quantification of TRF2 intensities in U2OS cells over-expressing empty vector (EV) or FLAG-tagged FAM111B variants wild-type (WT), protease-dead (PD) or HFP mutant (Q430P, QP), respectively. Green bars show averages of N = 3 experiments, with at least 50 cells per sample scored in each experiment. K-W test was used in C, E and (F) .

Article Snippet: Primary antibodies were as follows: mouse anti-human BLM (sc-365753, Santa Cruz Biotechnology) at 1:250 (IF), mouse anti-BrdU (347580, Becton Dickinson) at 1:20 (Flow cytometry), rabbit anti-human FAM111B (HPA038637, Atlas Antibodies) at 1:1,000 (IB and IF), mouse anti-human Histone H3 (10799, Abcam) at 1:1,000 (IB), mouse anti-human Lamin A/C (sc-376248, Santa Cruz Biotechnology) at 1:1,000 (IB and IF), rabbit anti-human Lamin B1 (12987-1-AP, Proteintech Group) at 1:1,000 (IB and IF), rabbit anti-human NUP42 (16587-1-AP, Proteintech Group) at 1:1,000 (IB), mouse anti-human PCNA (sc-56, Santa Cruz Biotechnology) at 1:1,000 (IF), rabbit anti-human SEC13 (15397-1-AP, Proteintech Group) at 1:1,000 (IB), mouse anti-human TRF2 (NB100-56506, Novus Biologicals) at 1:1,000 (IF), mouse anti-human αTubulin (T6074, Sigma) at 1:10,000 (WB) or 1:1,000 (IF).

Techniques: Knock-Out, Staining, Expressing, Plasmid Preparation, Mutagenesis

Journal: Molecular Biology of the Cell

Article Title: Telomeric DNA Mediates De Novo PML Body Formation

doi: 10.1091/mbc.E09-04-0309

Figure Lengend Snippet: Endogenous PML, Sp100 and Hausp accumulate at telomeric sites but not at centromeres in U2OS cells recovering from MMS treatment. (A) Immunofluorescence image of a U2OS cell treated with MMS, fixed and stained with anti-PML (green) and anti-TRF2 (red) antibodies. (B) Image of a U2OS cell that recovers from MMS treatment and is stained with antibodies against PML (green) and TRF2 (red). (C) Image of a HeLa cell that recovers from MMS treatment and is stained with antibodies against PML (green) and TRF2 (red). Arrows in B and C indicate the positions where PML colocalize or associate with TRF2 foci. (D) Localization of Sp100 at telomeric sites in a U2OS cell that recovers from MMS treatment. During recovery from MMS treatment, U2OS cells were fixed and stained with anti-Sp100 and anti-TRF2 antibodies. (E) Immunofluorescence image of a U2OS cell that recovers from MMS treatment. Sites where Hausp colocalize with telomeric DNA are indicated by arrows. (F) PML does not colocalize with centromeres in a U2OS cell that recovers from MMS treatment. After MMS treatment, U2OS cells were incubated in fresh medium, fixed and stained with anti-PML (green) and anti-CENPA (red) antibodies. All cell nuclei are counterstained with DAPI (blue).

Article Snippet: The following antibodies were used for immunofluorescence staining: mouse mAb 5E10 against PML (gift from R. van Driel, Amsterdam, The Netherlands), rabbit polyclonal antibody against PML (1130 directed against sequence: MEPAPARSPRPQQDP), rabbit polyclonal antibody against SP100 (ab1380, Chemicon, Temecula, CA), rabbit polyclonal antibody against Daxx (sc-7152, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit polyclonal antibody against Hausp (A300–033A, Bethyl Laboratories, Montgomery, TX), mouse mAb against TRF1 (ab10579–50, Abcam, Cambridge, MA), mouse mAb against TRF2 (IMG-124, Imgenex, San Diego, CA), human autoimmune serum against centromeres (Antibodies Incorporated, Davis, CA), rabbit polyclonal antibody against γH2AX (A300–081A, Bethyl Laboratories), rabbit polyclonal antibody against 53BP1 (NB100–304, Novus Biologicals, Littleton, CO), and rabbit polyclonal antibody against SMC5 (A300–236A, Bethyl Laboratories).

Techniques: Immunofluorescence, Staining, Incubation

Journal: Oncotarget

Article Title: Heregulin, a new interactor of the telosome/shelterin complex in human telomeres

doi:

Figure Lengend Snippet: Representative immunofluorescent images of MDA-MB-231 and Hs578T cells co-stained for endogenous HRGβ 2 (green) and A. RAP1 (red) or B. TRF2 (red) are shown ( n = 3). Merged images indicate co-localization (yellow). DAPI was used to visualize nuclear DNA (blue). Endogenous HRGβ 2 was immunoprecipitated from MDA-MB-231 or Hs578T nuclear extracts with pre-immune serum (PI) or a polyclonal anti-HRGβ 2 antibody (HRGβ 2 IP) in the absence (−) or presence (+) of a blocking HRGβ 2 peptide (BP). Precipitates were separated by SDS-PAGE, transferred to nitrocellulose, and probed with antibodies against A. RAP1 or B. TRF2. One representative immunoblot is shown ( n = 3).

Article Snippet: Cells were then stained with HRGβ 2 -, RAP1- and/or TRF2-specific antibodies (1:100 dilution of the anti-HRGβ 2 C20 antibody –sc-348-; Santa Cruz Biotech.

Techniques: Staining, Immunoprecipitation, Blocking Assay, SDS Page, Western Blot

Journal: Oncotarget

Article Title: Heregulin, a new interactor of the telosome/shelterin complex in human telomeres

doi:

Figure Lengend Snippet: A. Data reported by Li & Lange and O'Connor et al. originally suggested that RAP1 is a negative regulator of telomere length and that the BRCT, linker and/or Myb domains were required for this regulatory function. It was proposed that these RAP1 domains shouldinteract with a factor(s), i. e., either three different proteins, X , Y , and Z , or different domains of the same protein(s), which will be required fornegative regulation of telomere length. Our current mapping of the interacting regions between RAP1 and HRGβ 2 , together with the sequencing data from the Y2H screen, reveal that HRGβ 2 isa novel interactor of the telosome/shelterin complex through its interaction downstream of the BRCT domain of RAP1, likely between amino acids 127 and 259. HRGβ 2 should be viewed as one of the unknown negative regulators of telomere length recruited by the linker domain of RAP1 that, in addition, simultaneouslyinteracts with the RAP1 partner TRF2. B. The delineation of the 6 telomeric proteins (TRF1, TRF2, TIN2, RAP1, TPP1, and POT1) and their associated partners has provided the basis forconstructing an interaction map of telomere regulators in mammalian cells, the so-called telomere interactome [ – ], which represents the molecular machinery that controls mammalian telomeres and enables the integration of the various signaling pathways that regulate telomeres with other cellular interactomes. At the core ofthe telomere interactome is the telosome/shelterin complex, which can recruit a multitude of factors through various telosome subunits (e. g., RAP1) and mostly through protein hubs, which mediate multiple interactions with other proteins in the telomere interactome consistent with their key roles in telomere control (e. g., TRF2). Because TRF2 directly binds telomere DNA and recruits different proteins that are involved not only in cell cycle, DNA repair and recombination but also in end protection (e. g., RAP1) [ – , ], the ability of HRGβ 2 to interact with RAP1 and TRF2 strongly suggest that HRGβ 2 may significantly alter the protein-protein interaction within the telomere interactome to regulate telomere function and dynamics in normal development or in diseased cells, such as cancer and aging cells. Whether the RAP1-HRGβ 2 -TRF2 interaction actually determines whether the telomerase complex can be recruited or displaced from the core of the telosome/shelterin complex and/or whether TRF2 can efficiently remodel the telomere end into t-loopstructures , which sequester and protect the ends of chromosomes, remains to be determined.

Article Snippet: Cells were then stained with HRGβ 2 -, RAP1- and/or TRF2-specific antibodies (1:100 dilution of the anti-HRGβ 2 C20 antibody –sc-348-; Santa Cruz Biotech.

Techniques: Sequencing, Protein-Protein interactions, Control

Journal: Experimental & Molecular Medicine

Article Title: SUMOylation of TP53INP1 is involved in miR-30a-5p-regulated heart senescence

doi: 10.1038/s12276-024-01347-3

Figure Lengend Snippet: a qRT‒PCR analysis of miR-30a-5p expression in the heart tissues of 3- and 18-month-old WT mice ( n = 6 in each group). Representative RNA-FISH images ( b ) and analysis ( c ) of miR-30a-5p levels in heart tissues of 3- and 18-month-old WT mice ( n = 4 in each group). d qRT‒PCR analysis of miR-30a-5p levels in the hearts of vehicle- or D-gal-treated WT mice ( n = 5 in each group). Representative RNA-FISH images ( e ) and analysis ( f ) of miR-30a-5p levels in the heart tissues of vehicle- or D-gal-induced mice ( n = 3 in each group). Representative western blotting images ( g ) and analysis ( h ) of p53 and p21 expression in the heart tissues of 3- and 18-month-old WT mice ( n = 4 in each group). Representative western blotting images ( i ) and analysis ( j ) of p53 and p21 protein levels in the heart tissues of the vehicle- or D-gal-treated mice ( n = 4 in each group). k qRT‒PCR analysis of miR-30a-5p expression in the blood of 3- or 18-month-old mice ( n = 4 in both groups). l qRT‒PCR analysis of miR-30a-5p expression in NRCMs and NRCFs ( n = 6 in each group). m qRT‒PCR analysis of miR-30a-5p expression in 5- and 10-day-treated NRCMs ( n = 9 in each group). n qRT‒PCR analysis of miR-30a-5p expression in vehicle- and D-gal-induced NRCMs ( n = 10 in each group). The data are presented as the means ± standard errors. Statistical significance was assessed using t tests. WT wild-type, qRT‒PCR quantitative real-time polymerase chain reaction, RNA‒FISH RNA‒fluorescence in situ hybridization, veh vehicle, D‒gal D‒galactose, NRCMs neonatal rat cardiomyocytes, NRCFs neonatal rat cardiac fibroblasts.

Article Snippet: The primary antibodies used for western blotting were mouse monoclonal anti-p53 (Cat# Ab26, Abcam, Cambridge, UK), rabbit polyclonal anti-p21 (Cat# 8248-1-AP) and mouse monoclonal anti-TERF2 (Cat# 66893-1-Ig, both ProteinTech, Wuhan, China), rabbit polyclonal anti-TP53INP1 (Cat# OM204116, OmniMabs, Alhambra, CA, USA), mouse monoclonal anti-SUMO1 (Cat# sc-5308, Santa Cruz, Dallas, Texas, USA), rabbit polyclonal anti-TERT (Cat# DF7129), rabbit polyclonal anti-DDIT4 (Cat# 10638-1-AP, Proteintech, Wuhan, China), and rabbit polyclonal anti-SENP1 (Cat# AF0275, both Affinity Bioscience, Nanjing, China).

Techniques: Expressing, Western Blot, Real-time Polymerase Chain Reaction, In Situ Hybridization

Journal: Experimental & Molecular Medicine

Article Title: SUMOylation of TP53INP1 is involved in miR-30a-5p-regulated heart senescence

doi: 10.1038/s12276-024-01347-3

Figure Lengend Snippet: Representative ECG images ( a ) and analysis ( b ) of LVEF and LVFS in 2- and 18-month-old WT and KO mice ( n = 7 in each group). Representative images of immunofluorescence staining ( c ) and analysis ( d ) of γH2AX in heart tissues of 2- and 18-month-old WT and KO mice (scale bars=200 μm; n = 3 in each group). e qRT‒PCR analysis of telomere length in heart tissues of 2- and 18-month-old WT and KO mice ( n = 6 in each group). Representative western blotting images ( f ) and analysis ( g ) of the expression of p21 ( n = 5 in each group) and p53 ( n = 4 in each group) in the heart tissues of 2- and 18-month-old WT and KO mice. qRT‒PCR analysis of myocardial IL-1β ( h , n = 5 in each group) and IL-6 ( i , n = 4 in each group) levels. Representative Picrosirius Red (scale bars = 50 μm) and wheat germ agglutinin (WGA, scale bars = 100 μm) staining images ( j ) and analysis ( k ) of the hearts of 2- and 18-month-old WT and KO mice ( n = 5 in each group). The data are presented as the means ± standard errors. Statistical significance was assessed using one-way ANOVA. ECG echocardiography, WT wild-type, KO knockout, LVEF left ventricular ejection fraction, LVFS left ventricular fractional shortening, qRT‒PCR quantitative real-time polymerase chain reaction, γH2AX H2A histone family member X, WGA wheat germ agglutinin.

Article Snippet: The primary antibodies used for western blotting were mouse monoclonal anti-p53 (Cat# Ab26, Abcam, Cambridge, UK), rabbit polyclonal anti-p21 (Cat# 8248-1-AP) and mouse monoclonal anti-TERF2 (Cat# 66893-1-Ig, both ProteinTech, Wuhan, China), rabbit polyclonal anti-TP53INP1 (Cat# OM204116, OmniMabs, Alhambra, CA, USA), mouse monoclonal anti-SUMO1 (Cat# sc-5308, Santa Cruz, Dallas, Texas, USA), rabbit polyclonal anti-TERT (Cat# DF7129), rabbit polyclonal anti-DDIT4 (Cat# 10638-1-AP, Proteintech, Wuhan, China), and rabbit polyclonal anti-SENP1 (Cat# AF0275, both Affinity Bioscience, Nanjing, China).

Techniques: Immunofluorescence, Staining, Western Blot, Expressing, Knock-Out, Real-time Polymerase Chain Reaction

Journal: Experimental & Molecular Medicine

Article Title: SUMOylation of TP53INP1 is involved in miR-30a-5p-regulated heart senescence

doi: 10.1038/s12276-024-01347-3

Figure Lengend Snippet: a Schematic of the experimental timeline of D-gal-induced aging in WT and KO mice. Representative ECG images ( b ) and analysis ( c ) of the LVEF and LVFS of Veh- or D-gal-induced WT and KO mice ( n = 7 in each group). Representative immunofluorescence staining ( d ) and analysis ( e ) of γH2AX in heart tissues of Veh- or D-gal-induced WT and KO mice ( n = 4 in each group, scale bars = 200 μm). f qRT‒PCR analysis of telomere length in heart tissues from Veh- or D-gal-induced WT and KO mice ( n = 6 in each group). Representative western blotting images ( g ) and analysis ( h ) of p53 and p21 expression in heart tissues of Veh- or D-gal-induced WT and KO mice ( n = 4 in each group). Representative WGA staining images ( i ) and analysis ( j ) of the hearts of Veh- or D-gal-induced WT and KO mice ( n = 5 in each group; scale bars = 100 μm). k qRT‒PCR analysis of myocardial IL-1β ( n = 3 in each group) and IL-6 ( n = 4 in each group) levels in Veh- or D-gal-induced WT and KO mice. The data are presented as the means ± standard errors. Statistical significance was assessed using one-way ANOVA. ECG echocardiography, WT wild-type, KO knockout, LVEF left ventricular ejection fraction, LVFS left ventricular fractional shortening, D-gal D-galactose, qRT‒PCR quantitative real‒time polymerase chain reaction, γH2AX H2A histone family member X, Veh vehicle, WGA wheat germ agglutinin.

Article Snippet: The primary antibodies used for western blotting were mouse monoclonal anti-p53 (Cat# Ab26, Abcam, Cambridge, UK), rabbit polyclonal anti-p21 (Cat# 8248-1-AP) and mouse monoclonal anti-TERF2 (Cat# 66893-1-Ig, both ProteinTech, Wuhan, China), rabbit polyclonal anti-TP53INP1 (Cat# OM204116, OmniMabs, Alhambra, CA, USA), mouse monoclonal anti-SUMO1 (Cat# sc-5308, Santa Cruz, Dallas, Texas, USA), rabbit polyclonal anti-TERT (Cat# DF7129), rabbit polyclonal anti-DDIT4 (Cat# 10638-1-AP, Proteintech, Wuhan, China), and rabbit polyclonal anti-SENP1 (Cat# AF0275, both Affinity Bioscience, Nanjing, China).

Techniques: Immunofluorescence, Staining, Western Blot, Expressing, Knock-Out, Polymerase Chain Reaction

Journal: Experimental & Molecular Medicine

Article Title: SUMOylation of TP53INP1 is involved in miR-30a-5p-regulated heart senescence

doi: 10.1038/s12276-024-01347-3

Figure Lengend Snippet: a Schematic of the experimental design of D-gal-induced aging in WT mice transfected with AAV9-NC-sponge or AAV9-miR-30a-5p-sponge. Representative ECG images ( b ) and analysis ( c ) of the LVEF and LVFS of Veh- or D-gal-induced mice transfected with AAV9-cTnT-NC-sponge or AAV9-cTnT-miR-30a-5p-sponge ( n = 6 in each group). Representative immunofluorescence staining ( d ) and analysis ( e ) of γH2AX in heart tissues of Veh- or D-gal-induced aging in mice treated with AAV9-cTnT-NC-sponge or AAV9-cTnT-miR-30a-5p-sponge ( n = 6 in each group, scale bars = 100 μm). f qRT‒PCR analysis of telomere length in heart tissues of Veh- or D-gal-induced aging in mice treated with AAV9-cTnT-NC-sponge or AAV9-cTnT-miR-30a-5p-sponge ( n = 6 in each group). Representative WGA staining images ( g ) and analysis ( h ) of the hearts of Veh- or D-gal-induced aging in mice treated with AAV9-cTnT-NC-sponge or AAV9-cTnT-miR-30a-5p-sponge ( n = 5 in each group, scale bars = 100 μm). i qRT‒PCR analysis of myocardial IL-1β and IL-6 levels in Veh- or D-gal-induced aging in mice treated with AAV9-cTnT-NC-sponge or AAV9-cTnT-miR-30a-5p-sponge ( n = 3 in each group). Representative SA-β-gal staining images ( j ) and analysis ( k ) of NRCMs transfected with control inhibitor or miR-30a-5p inhibitor cultured for 5 or 10 days ( n = 3 in each group, scale bars = 100 μm). l qRT‒PCR analysis of telomere length in NRCMs cultured for 5 or 10 days and transfected with control inhibitor or miR-30a-5p inhibitor ( n = 6 in each group). Representative western blotting images ( m ) and analysis ( n ) of p53 ( n = 4 in each group) and p21 ( n = 3 in each group) expression in NRCMs transfected with the control inhibitor or the miR-30a-5p inhibitor and cultured for 5 or 10 days. The data are presented as the means ± standard errors. Statistical significance was assessed using one-way ANOVA. WT wild-type, LVEF left ventricular ejection fraction, LVFS left ventricular fractional shortening, Veh vehicle, D-gal D-galactose, ECG echocardiography, qRT‒PCR quantitative real-time polymerase chain reaction, γH2AX H2A histone family member X, NRCMs neonatal rat cardiomyocytes.

Article Snippet: The primary antibodies used for western blotting were mouse monoclonal anti-p53 (Cat# Ab26, Abcam, Cambridge, UK), rabbit polyclonal anti-p21 (Cat# 8248-1-AP) and mouse monoclonal anti-TERF2 (Cat# 66893-1-Ig, both ProteinTech, Wuhan, China), rabbit polyclonal anti-TP53INP1 (Cat# OM204116, OmniMabs, Alhambra, CA, USA), mouse monoclonal anti-SUMO1 (Cat# sc-5308, Santa Cruz, Dallas, Texas, USA), rabbit polyclonal anti-TERT (Cat# DF7129), rabbit polyclonal anti-DDIT4 (Cat# 10638-1-AP, Proteintech, Wuhan, China), and rabbit polyclonal anti-SENP1 (Cat# AF0275, both Affinity Bioscience, Nanjing, China).

Techniques: Transfection, Immunofluorescence, Staining, Control, Cell Culture, Western Blot, Expressing, Real-time Polymerase Chain Reaction

Journal: Experimental & Molecular Medicine

Article Title: SUMOylation of TP53INP1 is involved in miR-30a-5p-regulated heart senescence

doi: 10.1038/s12276-024-01347-3

Figure Lengend Snippet: a Schematic of the experimental timeline of D-gal-induced aging in mice transfected with AAV9-cTnT-control (NC) or AAV9-cTnT-miR-30a-5p-overexpressing (OE) vectors. Representative ECG images ( b ) and analysis ( c ) of LVEF and LVFS in NC or OE WT mice induced with Veh or D-gal ( n = 6 in each group). d qRT‒PCR analysis of telomere length in the heart tissues of NC or OE WT mice induced with Veh or D-gal ( n = 6 in each group). Representative immunofluorescence staining ( e ) and analysis ( f ) of γH2AX in the heart tissues of NC or OE WT mice induced with Veh or D-gal ( n = 3 in each group; scale bars = 100 μm). Representative Picrosirius Red and WGA staining images ( g ) and analysis ( h and i ) of the hearts of NC or OE WT mice induced with Veh or D-gal ( n = 5 in each group; scale bars = 50 μm or 100 μm). qRT‒PCR analysis of myocardial IL-1β ( j ) and IL-6 ( k ) levels in NC or OE mice induced with Veh or D-gal ( n = 6 in each group). Representative SA-β-gal staining images ( l ) and analysis ( m ) of NRCMs cultured for 5 or 10 days and transfected with control or miR-30a-5p mimics ( n = 3 per group; scale bars = 100 μm). Representative SA-β-gal staining images ( n ) and analysis ( o ) of NRCMs transfected with control or miR-30a-5p mimics in the presence or absence of D-gal ( n = 3 per group, scale bars = 100 μm). p qRT‒PCR analysis of telomere length in NRCMs cultured for 5 or 10 days and transfected with control mimics or miR-30a-5p mimics ( n = 5 in each group). q qRT‒PCR analysis of telomere length in NRCMs transfected with control or miR-30a-5p mimics in the presence or absence of D-gal ( n = 8 in each group). Representative western blotting images ( r ) and analysis ( s ) of p53 ( n = 3 in each group) and p21 ( n = 4 in each group) expression in NRCMs cultured for 5 or 10 days and transfected with control or miR-30a-5p mimics. Representative western blotting images ( t ) and analysis ( u ) of p53 ( n = 3 in each group) and p21 ( n = 4 in each group) expression in NRCMs transfected with control or miR-30a-5p mimics in the presence or absence of D-gal. The data are presented as the means ± standard errors. Statistical significance was assessed using one-way ANOVA. NC control, OE overexpression, ECG echocardiography, LVEF left ventricular ejection fraction, LVFS left ventricular fractional shortening, D-gal D-galactose, WT wild-type, qRT‒PCR quantitative real-time polymerase chain reaction, γH2AX H2A histone family member X, β-gal β-galactosidase, Veh vehicle, NRCMs neonatal rat cardiomyocytes, SA-β-gal senescence-associated β-galactosidase.

Article Snippet: The primary antibodies used for western blotting were mouse monoclonal anti-p53 (Cat# Ab26, Abcam, Cambridge, UK), rabbit polyclonal anti-p21 (Cat# 8248-1-AP) and mouse monoclonal anti-TERF2 (Cat# 66893-1-Ig, both ProteinTech, Wuhan, China), rabbit polyclonal anti-TP53INP1 (Cat# OM204116, OmniMabs, Alhambra, CA, USA), mouse monoclonal anti-SUMO1 (Cat# sc-5308, Santa Cruz, Dallas, Texas, USA), rabbit polyclonal anti-TERT (Cat# DF7129), rabbit polyclonal anti-DDIT4 (Cat# 10638-1-AP, Proteintech, Wuhan, China), and rabbit polyclonal anti-SENP1 (Cat# AF0275, both Affinity Bioscience, Nanjing, China).

Techniques: Transfection, Control, Immunofluorescence, Staining, Cell Culture, Western Blot, Expressing, Over Expression, Real-time Polymerase Chain Reaction

Journal: Experimental & Molecular Medicine

Article Title: SUMOylation of TP53INP1 is involved in miR-30a-5p-regulated heart senescence

doi: 10.1038/s12276-024-01347-3

Figure Lengend Snippet: a Schematic of the experimental timeline of the D-gal-induced aging model in WT and KO mice transfected with AAV9-cTnT-control- or AAV9-cTnT-TP53INP1-RNAi. Representative ECG images ( b ) and analysis ( c ) of the LVEF and LVFS of D-gal-induced aged WT or KO mice transfected with AAV9-cTnT-control- or AAV9-cTnT-TP53INP1-RNAi ( n = 8 in each group). d qRT‒PCR analysis of telomere length in heart tissues of AAV9-control- or AAV9-TP53INP1-RNAi-treated WT or KO mice induced with D-gal ( n = 5 in each group). Representative immunofluorescence staining ( e ) and analysis ( f ) of γH2AX in heart tissues of AAV9-cTnT-control- or AAV9-cTnT-TP53INP1-RNAi-treated WT or KO mice induced with D-gal ( n = 3 in each group; scale bars = 100 μm). Representative western blotting images ( g ) and analysis ( h ) of TP53INP1 ( n = 7 in each group), p53 ( n = 7 in each group), and p21 ( n = 7 in each group) expression in heart tissues of AAV9-cTnT-control- or AAV9-cTnT-TP53INP1-RNAi-transfected WT or KO mice induced with D-gal. Representative WGA staining images ( i ) and analysis ( j ) of the hearts of AAV9-cTnT-control- or AAV9-cTnT-TP53INP1-RNAi-transfected WT or KO mice induced with D-gal ( n = 5 in each group, scale bars = 100 μm). k qRT‒PCR analysis of myocardial IL-1β ( n = 5 in each group) and IL-6 ( n = 6 in each group) levels in AAV9-cTnT-control- or AAV9-cTnT-TP53INP1-RNAi-transfected WT or KO mice induced with D-gal. The data are presented as the means ± standard errors. Statistical significance was assessed using one-way ANOVA. WT wild-type, KO knockout, ECG echocardiography, LVEF left ventricular ejection fraction, LVFS left ventricular fractional shortening, D-gal D-galactose, qRT‒PCR quantitative real-time polymerase chain reaction, γH2AX H2A histone family member X.

Article Snippet: The primary antibodies used for western blotting were mouse monoclonal anti-p53 (Cat# Ab26, Abcam, Cambridge, UK), rabbit polyclonal anti-p21 (Cat# 8248-1-AP) and mouse monoclonal anti-TERF2 (Cat# 66893-1-Ig, both ProteinTech, Wuhan, China), rabbit polyclonal anti-TP53INP1 (Cat# OM204116, OmniMabs, Alhambra, CA, USA), mouse monoclonal anti-SUMO1 (Cat# sc-5308, Santa Cruz, Dallas, Texas, USA), rabbit polyclonal anti-TERT (Cat# DF7129), rabbit polyclonal anti-DDIT4 (Cat# 10638-1-AP, Proteintech, Wuhan, China), and rabbit polyclonal anti-SENP1 (Cat# AF0275, both Affinity Bioscience, Nanjing, China).

Techniques: Transfection, Control, Immunofluorescence, Staining, Western Blot, Expressing, Knock-Out, Real-time Polymerase Chain Reaction

Journal: bioRxiv

Article Title: The APE2 nuclease is essential for DNA double strand break repair by microhomology-mediated end-joining

doi: 10.1101/2022.07.21.500989

Figure Lengend Snippet: A. Western Blot of Ku80, TRF2 and Actin in parental and XRCC5 KO TERF2 F/- upon treatment with 4OHT (1µM). B. Percentage of fused telomeres in XRCC5 WT and XRCC5 KO TERF2 F/- MEFs upon treatment with tamoxifen (4OHT, 1µM), DNA-PKcs-inhibitor (NU7441, 1µM) and PARP inhibitor (Olaparib, 20µM). 3 independent experiments. n=20 metaphases counted for each condition, each replica. Data are mean ± s.d. C. Experimental timeline for . sgNT: non-targeting small guide RNA. sgGOI: guide RNA targeting a gene of interest. D. Representative images of metaphase spreads analyzed in e. Red: DNA (DNA - DAPI), Green: telomere FISH. Scale bar: 5µm. E. Quantification of telomere fusions in XRCC5 KO TERF2 F/- MEFs transduced with iCas9 and the indicated sgRNA, and treated with doxycycline (1µg/ml), 4OHT (1µM), and PARPi (Olaparib, 20µM). 3 independent experiments. n=22 metaphases counted for each condition, each replica. Data are mean ± s.e.m. Statistical analyses: Grey: untreated vs. PARPi. Black: Untreated sgNT vs. untreated no guide or sgTarget. F. Quantification of telomere fusions in XRCC5 WT TERF2 F/- MEFs transduced with iCas9 and the indicated sgRNA, treated with doxycycline (1µg/ml) and 4OHT (1µM). 3 independent experiments. n=18 metaphases for each condition, each replica. Data are mean ± s.e.m. Statistical analysis for B, E and F: One-way ANOVA. ****p<0.0001.

Article Snippet: Antibodies used: BRCA1 (Cell Signaling, #9010T, diluted at 1:1000), PALB2 (generous gift from Jean-Yves Masson), Myc (Cell Signaling, #2276, diluted at 1:1000), TRF2 (Novus, #NB110-57130, diluted at 1:1000), Ku80 (Cell Signaling, #2753, diluted at 1:1000), ß-actin (Cell Signaling, #8457, diluted at 1:5000), secondary anti-Mouse-HRP (Cell Signaling, #70765, diluted at 1:3000), and secondary anti- Rabbit-HRP (Cell Signaling, #7074P2, diluted at 1:3000).

Techniques: Western Blot, Transduction

Journal: bioRxiv

Article Title: The APE2 nuclease is essential for DNA double strand break repair by microhomology-mediated end-joining

doi: 10.1101/2022.07.21.500989

Figure Lengend Snippet: A. Schematic of wild type and mutants APE2. Amino acids mutated or deleted are indicated for both human and mouse APE2. B. Western Blot of Myc-tag (mAPE2) and Actin for the indicated MEFs used in D. C. Experimental timeline for and : On day 0, Doxycycline treatment induces expression of Cas9 ( APEX2 knockout) and mAPE2 exogenous expression. On day 3, addition of 4OHT induces TERF2 Cre recombination and telomere deprotection. On Day 8, cells are briefly treated with Colcemid and metaphase spreads are prepared. D. Quantification of telomere fusion in XRCC5 KO TERF2 F/- MEFs transduced with iCas9, sg APEX2 #1, and the indicated mAPE2 construct, and treated with doxycycline (1µg/ml) and 4OHT (1µM). EV: empty vector. 3 independent experiments. 26 metaphases were counted in each condition, each replica. Data are mean ± s.e.m. E. Western Blot of Myc-tag (hAPE2) and Actin for the indicated HT1080s used in G. F. Experimental timeline for and . Cells were treated 3 days with Doxycycline to induce expression of Cas9 ( APEX2 knockout) and of the hAPE2 exogenous constructs prior to transfection with ISce1-BFP. 24 hours post-transfection, media was replaced (with Dox). Flow cytometry was performed 6 days after ISce1 transfection. G. MMEJ quantification using reporter from 2a in HT1080- APEX2 KO clone A2.4 complemented with indicated hAPE2. mCherry+ cells (MMEJ+) were scored in the BFP+ population (I-Sce1+). Values are normalized to WT. 4 independent experiments. Data are mean ± s.e.m. Statistical analysis for D and G: One-way ANOVA. Grey: vs empty vector, Black: vs WT. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: Antibodies used: BRCA1 (Cell Signaling, #9010T, diluted at 1:1000), PALB2 (generous gift from Jean-Yves Masson), Myc (Cell Signaling, #2276, diluted at 1:1000), TRF2 (Novus, #NB110-57130, diluted at 1:1000), Ku80 (Cell Signaling, #2753, diluted at 1:1000), ß-actin (Cell Signaling, #8457, diluted at 1:5000), secondary anti-Mouse-HRP (Cell Signaling, #70765, diluted at 1:3000), and secondary anti- Rabbit-HRP (Cell Signaling, #7074P2, diluted at 1:3000).

Techniques: Western Blot, Expressing, Knock-Out, Transduction, Construct, Plasmid Preparation, Transfection, Flow Cytometry

Journal: bioRxiv

Article Title: The APE2 nuclease is essential for DNA double strand break repair by microhomology-mediated end-joining

doi: 10.1101/2022.07.21.500989

Figure Lengend Snippet: A. Schematic of hAPE2 amino acid conservation. Two uncharacterized conserved regions are indicated. B. AlphaFold2 structure prediction of mAPE2. C. Schematic of APE2 CR1 and CR2 deletion mutants. Deleted amino acids are indicated for both human and mouse APE2. D. Quantification of telomere fusion in XRCC5 KO TERF2 F/- MEFs transduced with iCas9, sg APEX2 #1, and the indicated mAPE2 construct, and treated with doxycycline (1µg/ml) and 4OHT (1µM). EV = empty vector. 3 independent experiments. Data are mean ± s.e.m. Right: Western blot showing expression of the mAPE2 constructs. E. MMEJ quantification using the MMEJ repair reporter in HT1080- APEX2 KO clone A2.4 complemented with indicated hAPE2. mCherry+ cells (MMEJ+) were scored in the BFP+ population (I-Sce1+). Values are normalized to WT. 4 independent experiments. Data are mean ± s.e.m. Right: Western blot showing expression of the mAPE2 constructs. F-G. Recruitment of GFP-hAPE2 to sites of laser micro-irradiation (405 nm, 60% power) upon treatment with BrdU (10µM, 20h) in U2OS cells transfected with the indicated constructs. Data show mean of all cells analyzed over 3 independent experiments. In F, N=26 (WT, 1′CR1-NLS), 24 (1′CR1), or 29 (1′CR2) cells. In G, N=21 (WT) or 20 (CR1) cells. Statistical analysis for D and E: One-way ANOVA. Grey: vs empty vector, Black: vs WT. ***p<0.001, ****p<0.0001.

Article Snippet: Antibodies used: BRCA1 (Cell Signaling, #9010T, diluted at 1:1000), PALB2 (generous gift from Jean-Yves Masson), Myc (Cell Signaling, #2276, diluted at 1:1000), TRF2 (Novus, #NB110-57130, diluted at 1:1000), Ku80 (Cell Signaling, #2753, diluted at 1:1000), ß-actin (Cell Signaling, #8457, diluted at 1:5000), secondary anti-Mouse-HRP (Cell Signaling, #70765, diluted at 1:3000), and secondary anti- Rabbit-HRP (Cell Signaling, #7074P2, diluted at 1:3000).

Techniques: Transduction, Construct, Plasmid Preparation, Western Blot, Expressing, Irradiation, Transfection

Journal: Science Advances

Article Title: Telomerase promotes formation of a telomere protective complex in cancer cells

doi: 10.1126/sciadv.aav4409

Figure Lengend Snippet: ( A ) Representative fluorescent micrographs of HT1080 cells treated with two separate siRNAs targeting hTERT, with immunofluorescence for Hsp70-1 (red) and FISH for telomeres (green), and DAPI staining showing the nucleus (blue). Scale bars, 10 μm. White arrowheads indicate colocalizations between Hsp70-1 and telomeres (yellow), one of which is shown in the zoomed image on the far right. ( B ) Top: Relative hTERT mRNA expression measured by qRT-PCR after hTERT siRNA treatment of HT1080 cells (mean ± SE; n = 3 independent experiments). *** P < 0.001. Bottom: Quantitation of the colocalizations of Hsp70-1 and telomeres in HT1080 cells depleted of hTERT (mean ± SE; n = 3 independent experiments). **** P < 0.0001. ( C ) Representative fluorescent micrographs of GM639 cells sorted for cells overexpressing GFP-tagged WT hTERT or R3E/R6E hTERT, with immunofluorescence for Hsp70 (red), FISH for telomeres (green), and DAPI staining showing the nucleus (blue). Scale bars, 10 μm. White arrowheads indicate colocalizations between Hsp70 and telomeres (white), one of which is shown in the zoomed image on the far right. ( D ) Quantitation of colocalizations of Hsp70-1 and telomeres in GM639 cells overexpressing WT or R3E/R6E mutant hTERT (mean ± SE; n = 3 independent experiments). **** P < 0.0001. ( E ) Quantitation of colocalizations of Hsp70-1 and telomeres in GM639 cells overexpressing either D712A hTERT or ZNF827 (mean ± SE; n = 3 independent experiments). **** P < 0.0001. ( F ) PLA for detection of Hsp70-1 and TRF2 interactions (red) in HT1080 cells with and without hTERT overexpression. See also the Supplementary Materials, fig. S6.

Article Snippet: Antibodies used for Western blotting include anti-hTERT (goat polyclonal, catalog no. SC7215, 1:1000; Santa Cruz Biotechnology), anti-pS1981 ATM (rabbit polyclonal, catalog no. AB81292, 1:1000; Abcam), anti-ATM (rabbit polyclonal, catalog no. AB32420, 1:2000; Abcam), anti-pT68 Chk2 (rabbit polyclonal, catalog no. 2661, 1:1000; Cell Signaling), anti-Chk2 (mouse monoclonal, catalog no. 05-649,1:1000; Merck Millipore), anti-Hsp70/Hsp72 2H9 (mouse monoclonal, catalog no. MABE1130, 1:2000; specific for Hsp70-1; Merck Millipore), anti-Hsp70 (rabbit polyclonal, catalog no. ab79852, 1:10,000; Abcam), anti-TRF2 (rabbit polyclonal, catalog no. NB110-57130, 1:400; Novus), anti-Myc tag (71D10) (rabbit polyclonal, catalog no. 2278, 1:1000; Cell Signaling), anti-DYKDDDDK Tag (rabbit polyclonal, catalog no. 2368S, 1:1000; Cell Signaling), anti-vinculin (mouse monoclonal, catalog no. V9131, 1:10000; Sigma-Aldrich), and anti-actin (rabbit polyclonal, catalog no. A2103, 1:10000; Sigma-Aldrich).

Techniques: Immunofluorescence, Staining, Expressing, Quantitative RT-PCR, Quantitation Assay, Mutagenesis, Over Expression

Journal: Science Advances

Article Title: Telomerase promotes formation of a telomere protective complex in cancer cells

doi: 10.1126/sciadv.aav4409

Figure Lengend Snippet: ( A ) Quantitation of the percentage of γ-H2AX–associated telomeres from meta-TIF assays in GM639 cells (mean ± SE; n = 3 independent experiments) with or without overexpression of TRF2. *** P = 0.0002. ( B ) Left: Representative Western blot showing transient overexpression of Myc-DDK–tagged Apollo (60 kDa) in HT1080 cells. Actin is used as a loading control. Right: PLA conducted for detection of Apollo-TRF2 interactions (red) in HT1080 cells with myc-Apollo overexpression. An anti-Myc tag antibody was used for Apollo detection. ( C ) PLA for detection of Apollo-Hsp70 interactions (red) in HT1080 cells overexpressing Apollo with and without hTERT overexpression. An anti-Myc tag antibody was used for Apollo detection. ( D ) Immunoprecipitation using antibodies against Myc tag (mouse antibody), Hsp70-1 (mouse), and IgG control (mouse) from lysates of 293T cells overexpressing Myc-DDK (FLAG)–tagged Apollo. Input (2 × 10 4 cell equivalents) and eluates were visualized on Western blots probed with antibodies against DDK (FLAG) tag (rabbit) and Hsp70 (rabbit). Volumes of eluate loaded correspond to the following cell equivalents: 5 × 10 5 (Apollo eluate on Apollo blot and Hsp70 eluate on Hsp70 blot), 1 × 10 6 (IgG control), 3 × 10 6 (Hsp70 eluate on Apollo blot), and 4 × 10 6 (Apollo eluate on Hsp70 blot). ( E ) Representative fluorescent micrographs (left) and quantitation of normalized colocalizations per cell (right) (mean ± SE; n = 3 independent experiments) of HT1080 cells overexpressing Myc-DDK–tagged Apollo and the indicated siRNAs, with immunofluorescence for Myc tag (red) and TRF2 (green), and DAPI staining showing the nucleus (blue). Scale bars, 10 μm. White arrowheads indicate colocalizations between Apollo and TRF2 (white). * P = 0.038, ** P < 0.01. ( F ) Quantitation of the percentage of γ-H2AX–associated telomeres from meta-TIF assays in GM639 cells with knock-down of DCLRE1B (Apollo), with and without overexpression of D712A hTERT (mean ± SE; n = 3). ** P = 0.0056. Note that the depletion of Apollo in GM639 cells, which have a high basal level of meta-TIFs, did not further increase telomere deprotection, suggesting that this cell line is unable to tolerate higher levels of telomere dysfunction. ( G ) Representative fluorescent micrographs of GM639 cells overexpressing WT hTERT with immunofluorescence for Hsp70-1 (red) and TRF2 (green), and DAPI staining showing the nucleus (blue). Scale bars, 10 μm. White arrowheads indicate colocalizations between Hsp70-1 and TRF2 (yellow), one of which is shown in the zoomed image on the right. ( H ) Quantitation of the colocalizations of Hsp70-1 and TRF2 immunofluorescence in GM639 cells shown in (G) (mean ± SE; n = 3). **** P < 0.0001. ( I ) Left: Examples of normal or aberrant telomere staining on chromosomes from metaphase spreads of HT1080 cells, with FISH against telomeres (green) and centromeres (red). Right: Quantitation of numbers of “fragile telomeres” per metaphase in HT1080 cells depleted of Hsp70 (siHSPA1A + siHSPA1B), hTERT, or Apollo (mean ± SE; n = 40 metaphases counted from each of three independent experiments). * P = 0.02, **** P < 0.0001. See also the Supplementary Materials, fig. S7.

Article Snippet: Antibodies used for Western blotting include anti-hTERT (goat polyclonal, catalog no. SC7215, 1:1000; Santa Cruz Biotechnology), anti-pS1981 ATM (rabbit polyclonal, catalog no. AB81292, 1:1000; Abcam), anti-ATM (rabbit polyclonal, catalog no. AB32420, 1:2000; Abcam), anti-pT68 Chk2 (rabbit polyclonal, catalog no. 2661, 1:1000; Cell Signaling), anti-Chk2 (mouse monoclonal, catalog no. 05-649,1:1000; Merck Millipore), anti-Hsp70/Hsp72 2H9 (mouse monoclonal, catalog no. MABE1130, 1:2000; specific for Hsp70-1; Merck Millipore), anti-Hsp70 (rabbit polyclonal, catalog no. ab79852, 1:10,000; Abcam), anti-TRF2 (rabbit polyclonal, catalog no. NB110-57130, 1:400; Novus), anti-Myc tag (71D10) (rabbit polyclonal, catalog no. 2278, 1:1000; Cell Signaling), anti-DYKDDDDK Tag (rabbit polyclonal, catalog no. 2368S, 1:1000; Cell Signaling), anti-vinculin (mouse monoclonal, catalog no. V9131, 1:10000; Sigma-Aldrich), and anti-actin (rabbit polyclonal, catalog no. A2103, 1:10000; Sigma-Aldrich).

Techniques: Quantitation Assay, Over Expression, Western Blot, Immunoprecipitation, FLAG-tag, Immunofluorescence, Staining

Journal: Science Advances

Article Title: Telomerase promotes formation of a telomere protective complex in cancer cells

doi: 10.1126/sciadv.aav4409

Figure Lengend Snippet: hTERT and Hsp70-1 engage in a positive feedback loop, in which hTERT induces expression of Hsp70-1, resulting in its own stabilization and proper folding. We have demonstrated that hTERT is also able to promote the localization of Hsp70-1 to deprotected telomeres, where it stabilizes the Apollo-TRF2 complex. This allows Apollo to engage in its known function of facilitating telomere replication. In this way, hTERT provides a protective function to telomeres and thereby avoids telomere damage-induced cell cycle arrest.

Article Snippet: Antibodies used for Western blotting include anti-hTERT (goat polyclonal, catalog no. SC7215, 1:1000; Santa Cruz Biotechnology), anti-pS1981 ATM (rabbit polyclonal, catalog no. AB81292, 1:1000; Abcam), anti-ATM (rabbit polyclonal, catalog no. AB32420, 1:2000; Abcam), anti-pT68 Chk2 (rabbit polyclonal, catalog no. 2661, 1:1000; Cell Signaling), anti-Chk2 (mouse monoclonal, catalog no. 05-649,1:1000; Merck Millipore), anti-Hsp70/Hsp72 2H9 (mouse monoclonal, catalog no. MABE1130, 1:2000; specific for Hsp70-1; Merck Millipore), anti-Hsp70 (rabbit polyclonal, catalog no. ab79852, 1:10,000; Abcam), anti-TRF2 (rabbit polyclonal, catalog no. NB110-57130, 1:400; Novus), anti-Myc tag (71D10) (rabbit polyclonal, catalog no. 2278, 1:1000; Cell Signaling), anti-DYKDDDDK Tag (rabbit polyclonal, catalog no. 2368S, 1:1000; Cell Signaling), anti-vinculin (mouse monoclonal, catalog no. V9131, 1:10000; Sigma-Aldrich), and anti-actin (rabbit polyclonal, catalog no. A2103, 1:10000; Sigma-Aldrich).

Techniques: Expressing