wi38  (ATCC)


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    Structured Review

    ATCC wi38
    GBF-1 inhibition enhances adenovirus infection of melanoma cells. (A) Inhibition of GBF-1 by GCA enhances HAdV-C5-dE1_GFP infection of M950822 and M980928 melanoma cells but not normal human <t>WI38</t> fibroblasts. Cells were preincubated with DMSO or GCA for
    Wi38, supplied by ATCC, used in various techniques. Bioz Stars score: 97/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/wi38/product/ATCC
    Average 97 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
    wi38 - by Bioz Stars, 2022-09
    97/100 stars

    Images

    1) Product Images from "Chemical Induction of Unfolded Protein Response Enhances Cancer Cell Killing through Lytic Virus Infection"

    Article Title: Chemical Induction of Unfolded Protein Response Enhances Cancer Cell Killing through Lytic Virus Infection

    Journal: Journal of Virology

    doi: 10.1128/JVI.02156-14

    GBF-1 inhibition enhances adenovirus infection of melanoma cells. (A) Inhibition of GBF-1 by GCA enhances HAdV-C5-dE1_GFP infection of M950822 and M980928 melanoma cells but not normal human WI38 fibroblasts. Cells were preincubated with DMSO or GCA for
    Figure Legend Snippet: GBF-1 inhibition enhances adenovirus infection of melanoma cells. (A) Inhibition of GBF-1 by GCA enhances HAdV-C5-dE1_GFP infection of M950822 and M980928 melanoma cells but not normal human WI38 fibroblasts. Cells were preincubated with DMSO or GCA for

    Techniques Used: Inhibition, Infection

    2) Product Images from "RECQL5 cooperates with Topoisomerase II alpha in DNA decatenation and cell cycle progression"

    Article Title: RECQL5 cooperates with Topoisomerase II alpha in DNA decatenation and cell cycle progression

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr844

    RECQL5 depletion is associated with slow proliferation rate, arrest in G2/M phase, and late S-phase cycling defects. ( A ) Quantitative RT–PCR analysis of the Recql5 mRNA levels normalized to endogenous GAPDH, in HeLa shScrambled, shRECQL5-1 ( n = 3) P = 0.0006 and shRECQL5-2 ( n = 3) P = 0.0002, Student's t -test. ( B ) Methylene blue stained colonies of HeLa shScrambled and HeLa shRECQL5-2. Growth assays were performed with ( C ) HeLa, ( D ) WI38 or (E) HCT116 using shScrambled, shRECQL5-1 and shRECQL5-2 as described in ‘Materials and Methods’ section. Error bars represent ±SD, n = 3. The non-linear fit was calculated and R square ( Y = Y 0 * e k * X ) values are 0.989 for shScrambled, 0.982 for shRECQL5-1 and 0.7416 for shRECQL5-2 in HeLa, 0.983 for shScrambled, 0.927 for shRECQL5-1 and 0.25 for shRECQL5-2 for WI38 experiments and 0.995 for shScrambled, 0.993 for shRECQL5-1 and 0.976 for shRECQL5-2 in HCT 116 experiments. Graphical representation of the doubling time calculated as an average of two independent growth assays. Error bars represent ±SD, n = 6 for each sample, each day; *** P
    Figure Legend Snippet: RECQL5 depletion is associated with slow proliferation rate, arrest in G2/M phase, and late S-phase cycling defects. ( A ) Quantitative RT–PCR analysis of the Recql5 mRNA levels normalized to endogenous GAPDH, in HeLa shScrambled, shRECQL5-1 ( n = 3) P = 0.0006 and shRECQL5-2 ( n = 3) P = 0.0002, Student's t -test. ( B ) Methylene blue stained colonies of HeLa shScrambled and HeLa shRECQL5-2. Growth assays were performed with ( C ) HeLa, ( D ) WI38 or (E) HCT116 using shScrambled, shRECQL5-1 and shRECQL5-2 as described in ‘Materials and Methods’ section. Error bars represent ±SD, n = 3. The non-linear fit was calculated and R square ( Y = Y 0 * e k * X ) values are 0.989 for shScrambled, 0.982 for shRECQL5-1 and 0.7416 for shRECQL5-2 in HeLa, 0.983 for shScrambled, 0.927 for shRECQL5-1 and 0.25 for shRECQL5-2 for WI38 experiments and 0.995 for shScrambled, 0.993 for shRECQL5-1 and 0.976 for shRECQL5-2 in HCT 116 experiments. Graphical representation of the doubling time calculated as an average of two independent growth assays. Error bars represent ±SD, n = 6 for each sample, each day; *** P

    Techniques Used: Quantitative RT-PCR, Staining

    3) Product Images from "MECHANISM FOR NICOTINE-INDUCED UP-REGULATION OF WNT SIGNALING IN HUMAN ALVEOLAR INTERSTITIAL FIBROBLASTS"

    Article Title: MECHANISM FOR NICOTINE-INDUCED UP-REGULATION OF WNT SIGNALING IN HUMAN ALVEOLAR INTERSTITIAL FIBROBLASTS

    Journal: Experimental lung research

    doi: 10.3109/01902148.2010.490288

    Evidence for Protein Kinase C activation on nicotine treatement of WI38 cells
    Figure Legend Snippet: Evidence for Protein Kinase C activation on nicotine treatement of WI38 cells

    Techniques Used: Activation Assay

    4) Product Images from "Targeting the tumor-promoting microenvironment in MET-amplified NSCLC cells with a novel inhibitor of pro-HGF activation"

    Article Title: Targeting the tumor-promoting microenvironment in MET-amplified NSCLC cells with a novel inhibitor of pro-HGF activation

    Journal: Oncotarget

    doi: 10.18632/oncotarget.18260

    Inhibition of pro-HGF activation overcomes fibroblast-mediated resistance to MET tyrosine kinase inhibition ( A) EBC-1 cells were treated with JNJ38877605 (25 nM) alone or in the presence of conditioned medium (CM) from WI38 fibroblasts (FIB). CM was also prepared from fibroblasts with silenced HGF (HGF −/− FIB), or from fibroblasts cultured with HGF neutralizing antibody (α-HGF Ab) or SRI31215 (10 μM) as indicated. Cell viability was determined by CellTiter Glo ® 72 h after treatment. (B) H1993 cells were treated with JNJ38877605 (25 nM), fibroblast CM (FIB) and SRI31215 (10 μM) as indicated and cell viability was determined after 72 h. (C) Serum-starved EBC-1 cells were treated with JNJ38877605, recombinant HGF (100 nM), FIB and SRI31215 (10 μM) as indicated for 6 hours. Cell lysates were analyzed by immunoblotting for phospho- and total MET, EGFR, Gab1, AKT and ERK 1/2. *, p
    Figure Legend Snippet: Inhibition of pro-HGF activation overcomes fibroblast-mediated resistance to MET tyrosine kinase inhibition ( A) EBC-1 cells were treated with JNJ38877605 (25 nM) alone or in the presence of conditioned medium (CM) from WI38 fibroblasts (FIB). CM was also prepared from fibroblasts with silenced HGF (HGF −/− FIB), or from fibroblasts cultured with HGF neutralizing antibody (α-HGF Ab) or SRI31215 (10 μM) as indicated. Cell viability was determined by CellTiter Glo ® 72 h after treatment. (B) H1993 cells were treated with JNJ38877605 (25 nM), fibroblast CM (FIB) and SRI31215 (10 μM) as indicated and cell viability was determined after 72 h. (C) Serum-starved EBC-1 cells were treated with JNJ38877605, recombinant HGF (100 nM), FIB and SRI31215 (10 μM) as indicated for 6 hours. Cell lysates were analyzed by immunoblotting for phospho- and total MET, EGFR, Gab1, AKT and ERK 1/2. *, p

    Techniques Used: Inhibition, Activation Assay, Cell Culture, Recombinant

    5) Product Images from "Cell-specific Regulation of PTX3 by Glucocorticoid Hormones in Hematopoietic and Nonhematopoietic Cells *Cell-specific Regulation of PTX3 by Glucocorticoid Hormones in Hematopoietic and Nonhematopoietic Cells * S⃞"

    Article Title: Cell-specific Regulation of PTX3 by Glucocorticoid Hormones in Hematopoietic and Nonhematopoietic Cells *Cell-specific Regulation of PTX3 by Glucocorticoid Hormones in Hematopoietic and Nonhematopoietic Cells * S⃞

    Journal:

    doi: 10.1074/jbc.M805631200

    Regulation of PTX3 production by GC in WI38 fibroblast. A , a series of four experiments performed with Dex (10 -6 m ) and TNFα (20 ng/ml). Results refer to mean ± S.D. * , p
    Figure Legend Snippet: Regulation of PTX3 production by GC in WI38 fibroblast. A , a series of four experiments performed with Dex (10 -6 m ) and TNFα (20 ng/ml). Results refer to mean ± S.D. * , p

    Techniques Used:

    Induction of PTX3 mRNA by GC in fibroblasts and endothelial cells. WI38 fibroblasts ( left ) and HUVEC ( right ) were used. WI38 and HUVEC mRNA were obtained after 5 h of stimulation with TNFα (20 ng/ml), IL-1β (20 ng/ml), Dex (10 -6 m ),
    Figure Legend Snippet: Induction of PTX3 mRNA by GC in fibroblasts and endothelial cells. WI38 fibroblasts ( left ) and HUVEC ( right ) were used. WI38 and HUVEC mRNA were obtained after 5 h of stimulation with TNFα (20 ng/ml), IL-1β (20 ng/ml), Dex (10 -6 m ),

    Techniques Used:

    GC·GR complexes directly activate PTX3 transcription by GR binding to PTX3 promoter. Dex (10 -6 m ), TNFα (20 ng/ml), and LPS (100 ng/ml) are used. A , ChIP analysis in WI38. Polyclonal RNA pol II ( top ) and GR ( bottom ) antibodies are
    Figure Legend Snippet: GC·GR complexes directly activate PTX3 transcription by GR binding to PTX3 promoter. Dex (10 -6 m ), TNFα (20 ng/ml), and LPS (100 ng/ml) are used. A , ChIP analysis in WI38. Polyclonal RNA pol II ( top ) and GR ( bottom ) antibodies are

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation

    6) Product Images from "NKG2D ligands mediate immunosurveillance of senescent cells"

    Article Title: NKG2D ligands mediate immunosurveillance of senescent cells

    Journal: Aging (Albany NY)

    doi:

    Senescent cells upregulate MICA and ULBP2 on the cell surface ( A ) ImageStream analysis demonstrates higher expression levels of MICA and ULBP2 on the cell surface membrane of DIS IMR-90 fibroblasts (Senescent) compared to control (Growing) cells. ( B ) Quantification of total intensity and total membrane intensity indicate higher levels of MICA and ULBP2 in DIS IMR-90 cells compared to growing (control) cells. ( C ) Representative immunofluorescence staining of MICA and ULBP2 performed on non-permeabilized cells demonstrates higher expression levels of these proteins on the cell surface membrane in DIS WI38 and BJ cells compared to growing (control) cells. Scale bar: 50μm. MICA and ULBP2 are shown in red. DAPI is shown in blue. Data presented as mean with S.E.M of three independent experiments. Two-tailed t-test *P
    Figure Legend Snippet: Senescent cells upregulate MICA and ULBP2 on the cell surface ( A ) ImageStream analysis demonstrates higher expression levels of MICA and ULBP2 on the cell surface membrane of DIS IMR-90 fibroblasts (Senescent) compared to control (Growing) cells. ( B ) Quantification of total intensity and total membrane intensity indicate higher levels of MICA and ULBP2 in DIS IMR-90 cells compared to growing (control) cells. ( C ) Representative immunofluorescence staining of MICA and ULBP2 performed on non-permeabilized cells demonstrates higher expression levels of these proteins on the cell surface membrane in DIS WI38 and BJ cells compared to growing (control) cells. Scale bar: 50μm. MICA and ULBP2 are shown in red. DAPI is shown in blue. Data presented as mean with S.E.M of three independent experiments. Two-tailed t-test *P

    Techniques Used: Expressing, Immunofluorescence, Staining, Two Tailed Test

    MICA and ULBP2 expression is stably upregulated in senescent, but not quiescent cells Growing IMR-90, WI38 and BJ cells were treated with Etoposide (100μM) and the level of MICA and ULBP2 expression were assessed by RT-PCR. The expression of MICA ( A ) and ULBP2 ( B ) was elevated 24 hours following treatment and was maintained at higher levels throughout the indicated time points. Growing cells at day 0 served as control. ( C ) Growing cells were treated with a low concentration of Etoposide (10μM) to induce transient growth arrest and the level of MICA and ULBP2 expression were assessed by RT-PCR. ( D ) IMR-90 cells were grown to confluence and maintained for a further 7 days until cells became quiescent. Cell-cycle arrest induced by quiescence was not sufficient to elevate MICA and ULBP2 expression as assessed by RT-PCR and compared to growing and senescent cells. Data presented as mean with S.E.M of three independent experiments. Two-tailed t-test *P
    Figure Legend Snippet: MICA and ULBP2 expression is stably upregulated in senescent, but not quiescent cells Growing IMR-90, WI38 and BJ cells were treated with Etoposide (100μM) and the level of MICA and ULBP2 expression were assessed by RT-PCR. The expression of MICA ( A ) and ULBP2 ( B ) was elevated 24 hours following treatment and was maintained at higher levels throughout the indicated time points. Growing cells at day 0 served as control. ( C ) Growing cells were treated with a low concentration of Etoposide (10μM) to induce transient growth arrest and the level of MICA and ULBP2 expression were assessed by RT-PCR. ( D ) IMR-90 cells were grown to confluence and maintained for a further 7 days until cells became quiescent. Cell-cycle arrest induced by quiescence was not sufficient to elevate MICA and ULBP2 expression as assessed by RT-PCR and compared to growing and senescent cells. Data presented as mean with S.E.M of three independent experiments. Two-tailed t-test *P

    Techniques Used: Expressing, Stable Transfection, Reverse Transcription Polymerase Chain Reaction, Concentration Assay, Two Tailed Test

    Senescent cells upregulate NKG2D ligands RT-PCR analysis demonstrates a consistent up-regulation of MICA, ULBP1, and ULBP2 in DNA damage-induced senescent (DIS) ( A ), replicative senescent (RS) ( B ), and H-RAS v12 -mediated oncogene-induced senescent (OIS) ( C ) IMR-90 fibroblasts compared to growing (control) cells. Similarly, DIS WI38 ( D ) and BJ ( E ) fibroblasts and DIS hepatic stellate cells (HSCs) ( F ) also show an up-regulation of NKG2D ligands. The graphs represent the mean and the S.E.M of at least triplicate measurements from at least four independent experiments. Two-tailed t-test *P
    Figure Legend Snippet: Senescent cells upregulate NKG2D ligands RT-PCR analysis demonstrates a consistent up-regulation of MICA, ULBP1, and ULBP2 in DNA damage-induced senescent (DIS) ( A ), replicative senescent (RS) ( B ), and H-RAS v12 -mediated oncogene-induced senescent (OIS) ( C ) IMR-90 fibroblasts compared to growing (control) cells. Similarly, DIS WI38 ( D ) and BJ ( E ) fibroblasts and DIS hepatic stellate cells (HSCs) ( F ) also show an up-regulation of NKG2D ligands. The graphs represent the mean and the S.E.M of at least triplicate measurements from at least four independent experiments. Two-tailed t-test *P

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Two Tailed Test

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  • wi38  (ATCC)
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    ATCC wi38
    GBF-1 inhibition enhances adenovirus infection of melanoma cells. (A) Inhibition of GBF-1 by GCA enhances HAdV-C5-dE1_GFP infection of M950822 and M980928 melanoma cells but not normal human <t>WI38</t> fibroblasts. Cells were preincubated with DMSO or GCA for
    Wi38, supplied by ATCC, used in various techniques. Bioz Stars score: 94/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/wi38/product/ATCC
    Average 94 stars, based on 13 article reviews
    Price from $9.99 to $1999.99
    wi38 - by Bioz Stars, 2022-09
    94/100 stars
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    GBF-1 inhibition enhances adenovirus infection of melanoma cells. (A) Inhibition of GBF-1 by GCA enhances HAdV-C5-dE1_GFP infection of M950822 and M980928 melanoma cells but not normal human WI38 fibroblasts. Cells were preincubated with DMSO or GCA for

    Journal: Journal of Virology

    Article Title: Chemical Induction of Unfolded Protein Response Enhances Cancer Cell Killing through Lytic Virus Infection

    doi: 10.1128/JVI.02156-14

    Figure Lengend Snippet: GBF-1 inhibition enhances adenovirus infection of melanoma cells. (A) Inhibition of GBF-1 by GCA enhances HAdV-C5-dE1_GFP infection of M950822 and M980928 melanoma cells but not normal human WI38 fibroblasts. Cells were preincubated with DMSO or GCA for

    Article Snippet: A549 cells (American Type Culture Collection [ATCC]) are human lung epithelial carcinoma cells, HeLa-ATCC cells are cells of a HeLa cell clone obtained from the American Type Culture Collection, 911 cells are human embryonic retinoblasts containing base pairs 79 to 5789 of the HAdV-C5 genome , HEK293T cells are human embryonic kidney cells containing base pairs 1 to 4344 of the HAdV-C5 genome ( , ), and WI38 is a human diploid cell line derived from normal embryonic lung tissue (American Type Culture Collection).

    Techniques: Inhibition, Infection

    RECQL5 depletion is associated with slow proliferation rate, arrest in G2/M phase, and late S-phase cycling defects. ( A ) Quantitative RT–PCR analysis of the Recql5 mRNA levels normalized to endogenous GAPDH, in HeLa shScrambled, shRECQL5-1 ( n = 3) P = 0.0006 and shRECQL5-2 ( n = 3) P = 0.0002, Student's t -test. ( B ) Methylene blue stained colonies of HeLa shScrambled and HeLa shRECQL5-2. Growth assays were performed with ( C ) HeLa, ( D ) WI38 or (E) HCT116 using shScrambled, shRECQL5-1 and shRECQL5-2 as described in ‘Materials and Methods’ section. Error bars represent ±SD, n = 3. The non-linear fit was calculated and R square ( Y = Y 0 * e k * X ) values are 0.989 for shScrambled, 0.982 for shRECQL5-1 and 0.7416 for shRECQL5-2 in HeLa, 0.983 for shScrambled, 0.927 for shRECQL5-1 and 0.25 for shRECQL5-2 for WI38 experiments and 0.995 for shScrambled, 0.993 for shRECQL5-1 and 0.976 for shRECQL5-2 in HCT 116 experiments. Graphical representation of the doubling time calculated as an average of two independent growth assays. Error bars represent ±SD, n = 6 for each sample, each day; *** P

    Journal: Nucleic Acids Research

    Article Title: RECQL5 cooperates with Topoisomerase II alpha in DNA decatenation and cell cycle progression

    doi: 10.1093/nar/gkr844

    Figure Lengend Snippet: RECQL5 depletion is associated with slow proliferation rate, arrest in G2/M phase, and late S-phase cycling defects. ( A ) Quantitative RT–PCR analysis of the Recql5 mRNA levels normalized to endogenous GAPDH, in HeLa shScrambled, shRECQL5-1 ( n = 3) P = 0.0006 and shRECQL5-2 ( n = 3) P = 0.0002, Student's t -test. ( B ) Methylene blue stained colonies of HeLa shScrambled and HeLa shRECQL5-2. Growth assays were performed with ( C ) HeLa, ( D ) WI38 or (E) HCT116 using shScrambled, shRECQL5-1 and shRECQL5-2 as described in ‘Materials and Methods’ section. Error bars represent ±SD, n = 3. The non-linear fit was calculated and R square ( Y = Y 0 * e k * X ) values are 0.989 for shScrambled, 0.982 for shRECQL5-1 and 0.7416 for shRECQL5-2 in HeLa, 0.983 for shScrambled, 0.927 for shRECQL5-1 and 0.25 for shRECQL5-2 for WI38 experiments and 0.995 for shScrambled, 0.993 for shRECQL5-1 and 0.976 for shRECQL5-2 in HCT 116 experiments. Graphical representation of the doubling time calculated as an average of two independent growth assays. Error bars represent ±SD, n = 6 for each sample, each day; *** P

    Article Snippet: Cell lines HeLa, U2OS, WI38 and HCT116 were purchased from ATCC and grown according to ATCC protocols.

    Techniques: Quantitative RT-PCR, Staining

    Rad51-dependent subtelomere recombination triggered by forced resolution of cohesion is ATR and ChkI-dependent, POLD3-independent, and involves multiple subtelomeres. (A and B) Quantification of the frequency of TRF1.AA or TRF1.WT transfected late (PD50-53) WI38 mitotic cells (A) with cohered telomeres or (B) exhibiting subtelomere copying measured by FISH analysis with a 16p telo probe following treatment with the indicated inhibitors or siRNA. For TRF1.AA transfection (n=46). For TRF1.WT transfection, average of 2 independent experiments (n=25-50 cells each) ± SEM. (C) Immunoblot analysis of TRF1.WT-transfected, POLD3 siRNA-treated late (PD52) WI38 cell extracts. (D and E) Quantification of the frequency of WI38 TRF1.AA or TRF1.WT transfected late (PD55) mitotic cells (D) with cohered telomeres or (E) exhibiting subtelomere copying measured by dual FISH analysis with 13q and 16p telo probes. Average of two independent experiments (n=46-58 cells each) ± SEM. (F) FISH analysis of a WI38 TRF1.WT transfected late (PD55) mitotic cell exhibiting subtelomere copying using 13q (red) and 16p (green) telo probes. (G and H) Quantification of the frequency of TRF1.AA or TRF1.WT transfected late (PD52) WI38 mitotic cells (G) with cohered telomeres or (H) exhibiting subtelomere copying measured by dual FISH analysis with 13q and 4p telo probes. Average of two independent experiments (n=50 cells each) ± SEM. (I) FISH analysis of a TRF1.WT transfected late (PD52) WI38 mitotic cell exhibiting subtelomere copying using 13q (red) and 4p (green) telo probes. (F and I) DNA was stained with DAPI (blue). Scale bars represent 2 μm. *p ≤ 0.05, **p ≤ 0.01, (ns) not significant, Student’s unpaired t test. Source data are provided as a Source Data file.

    Journal: bioRxiv

    Article Title: Persistent telomere cohesion protects aged cells from premature senescence

    doi: 10.1101/2020.02.03.932145

    Figure Lengend Snippet: Rad51-dependent subtelomere recombination triggered by forced resolution of cohesion is ATR and ChkI-dependent, POLD3-independent, and involves multiple subtelomeres. (A and B) Quantification of the frequency of TRF1.AA or TRF1.WT transfected late (PD50-53) WI38 mitotic cells (A) with cohered telomeres or (B) exhibiting subtelomere copying measured by FISH analysis with a 16p telo probe following treatment with the indicated inhibitors or siRNA. For TRF1.AA transfection (n=46). For TRF1.WT transfection, average of 2 independent experiments (n=25-50 cells each) ± SEM. (C) Immunoblot analysis of TRF1.WT-transfected, POLD3 siRNA-treated late (PD52) WI38 cell extracts. (D and E) Quantification of the frequency of WI38 TRF1.AA or TRF1.WT transfected late (PD55) mitotic cells (D) with cohered telomeres or (E) exhibiting subtelomere copying measured by dual FISH analysis with 13q and 16p telo probes. Average of two independent experiments (n=46-58 cells each) ± SEM. (F) FISH analysis of a WI38 TRF1.WT transfected late (PD55) mitotic cell exhibiting subtelomere copying using 13q (red) and 16p (green) telo probes. (G and H) Quantification of the frequency of TRF1.AA or TRF1.WT transfected late (PD52) WI38 mitotic cells (G) with cohered telomeres or (H) exhibiting subtelomere copying measured by dual FISH analysis with 13q and 4p telo probes. Average of two independent experiments (n=50 cells each) ± SEM. (I) FISH analysis of a TRF1.WT transfected late (PD52) WI38 mitotic cell exhibiting subtelomere copying using 13q (red) and 4p (green) telo probes. (F and I) DNA was stained with DAPI (blue). Scale bars represent 2 μm. *p ≤ 0.05, **p ≤ 0.01, (ns) not significant, Student’s unpaired t test. Source data are provided as a Source Data file.

    Article Snippet: SV40 LT immortalization of WI38 cells For SV40 Large T antigen infection, amphotropic retroviruses were generated by transfecting 20 µg of pBabe-neoLargeTcDNA into Phoenix Amphotropic cells (ATCC) using calcium phosphate precipitation.

    Techniques: Transfection, Fluorescence In Situ Hybridization, Staining

    Loss of persistent telomere cohesion and subtelomere recombination accompany senescence onset. (A) FISH analysis of late IMR90 mitotic cells at PD48 and the final PD51 with a dual 13q arm (red) and telo (green) probe. (B) Quantification of the frequency of mitotic cells with cohered 13q arms (red) or telos (green). Average of two independent experiments (n=25-40 cells each) ± SEM. (C) Table of 13q FISH categories scored in IMR90 cells at PD48, 50 and 51. Average of two independent experiments (n=25-40 cells each) ± SEM. (D) FISH analysis of a final PD51 IMR90 mitotic cell exhibiting subtelomere, but not arm, copying using a dual 13q arm (red) and telo (green) probe. (E) Quantification of the frequency of mitotic cells exhibiting subtelomere or arm copying. Average of two independent experiments (n=25-40 cells each) ± SEM. (F) FISH analysis of late WI38 mitotic cells at PD52 and the final PD54 with a dual 13q arm (red) and telo (green) probe. (G) Quantification of the frequency of mitotic cells with cohered 13q arms (red) or telos (green). Average of two independent experiments (n=32-46 cells each) ± SEM. (H) Table of 13q FISH categories scored in late WI38 mitotic cells at PD 52, 53, and the final PD54. Average of two independent experiments (n=32-46 cells each) ± SEM. (I) FISH analysis of a final PD54 WI38 mitotic cell exhibiting subtelomere, but not arm, copying using a dual 13q arm (red) and telo (green) probe. (J) Quantification of the frequency of mitotic cells exhibiting subtelomere copying. Average of two independent experiments (n=32-46 cells each) ± SEM. (A, D, F, I) DNA was stained with DAPI (blue). Scale bars represent 2 μm. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, (ns) not significant, Student’s unpaired t test. Source data are provided as a Source Data file.

    Journal: bioRxiv

    Article Title: Persistent telomere cohesion protects aged cells from premature senescence

    doi: 10.1101/2020.02.03.932145

    Figure Lengend Snippet: Loss of persistent telomere cohesion and subtelomere recombination accompany senescence onset. (A) FISH analysis of late IMR90 mitotic cells at PD48 and the final PD51 with a dual 13q arm (red) and telo (green) probe. (B) Quantification of the frequency of mitotic cells with cohered 13q arms (red) or telos (green). Average of two independent experiments (n=25-40 cells each) ± SEM. (C) Table of 13q FISH categories scored in IMR90 cells at PD48, 50 and 51. Average of two independent experiments (n=25-40 cells each) ± SEM. (D) FISH analysis of a final PD51 IMR90 mitotic cell exhibiting subtelomere, but not arm, copying using a dual 13q arm (red) and telo (green) probe. (E) Quantification of the frequency of mitotic cells exhibiting subtelomere or arm copying. Average of two independent experiments (n=25-40 cells each) ± SEM. (F) FISH analysis of late WI38 mitotic cells at PD52 and the final PD54 with a dual 13q arm (red) and telo (green) probe. (G) Quantification of the frequency of mitotic cells with cohered 13q arms (red) or telos (green). Average of two independent experiments (n=32-46 cells each) ± SEM. (H) Table of 13q FISH categories scored in late WI38 mitotic cells at PD 52, 53, and the final PD54. Average of two independent experiments (n=32-46 cells each) ± SEM. (I) FISH analysis of a final PD54 WI38 mitotic cell exhibiting subtelomere, but not arm, copying using a dual 13q arm (red) and telo (green) probe. (J) Quantification of the frequency of mitotic cells exhibiting subtelomere copying. Average of two independent experiments (n=32-46 cells each) ± SEM. (A, D, F, I) DNA was stained with DAPI (blue). Scale bars represent 2 μm. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, (ns) not significant, Student’s unpaired t test. Source data are provided as a Source Data file.

    Article Snippet: SV40 LT immortalization of WI38 cells For SV40 Large T antigen infection, amphotropic retroviruses were generated by transfecting 20 µg of pBabe-neoLargeTcDNA into Phoenix Amphotropic cells (ATCC) using calcium phosphate precipitation.

    Techniques: Fluorescence In Situ Hybridization, Staining

    Subtelomere recombination leads to DNA damage and premature senescence in aged human fibroblasts. (A) Immunofluorescence analysis of Vector, TRF1.WT, or TRF1.AA infected late (PD51) WI38 cells with RAD51 antibody. (B) Quantification of the frequency of cells displaying > 5 RAD51 foci. Average of two independent experiments (n=100 cells each) ± SEM. (C) Immunofluorescence analysis of TRF1.WT-infected late (PD51) WI38 cells with Rad51 (green) and TRF2 (red) antibodies. (D) Quantification of the frequency of cells displaying ≥2 Rad51/TRF2 colocalizations. Average of two independent experiments (n=100 cells each) ± SEM. (E) Immunofluorescence analysis of Vector, TRF1.WT, or TRF1.AA infected late (PD51) WI38 cells with γH2AX (green) and 53BP1 (red) antibodies. (F) Quantification of the frequency of cells displaying > 5 γH2AX/53BP1 colocalizing foci. Average of three independent experiments (n=55-100 cells each) ± SEM. (G) Immunofluorescence analysis of TRF1.WT-infected late (PD51) WI38 cells with γH2AX (green) and TIN2 (red) antibodies. (H) Quantification of the frequency of cells with ≥4 γH2AX/TIN2 colocalizing foci. Average of two independent experiments (n=100 cells each) ± SEM. (I) Growth curve analysis of Vector, TRF1.WT, or TRF1.AA infected late (PD52) WI38 cells. Average of two independent experiments ± SEM. (J) SA-β-gal analysis of Vector, TRF1.WT, or TRF1.AA infected late (PD51) WI38 cells. Scale bar represent 100 μm. (K) Quantification of SA-β-gal positive cells. Average of two independent experiments (n=50-100 cells each) ± SEM. (L) Detection of senescence-associated heterochromatin foci (SAHF) in Vector, TRF1.WT, or TRF1.AA infected late (PD51) WI38. (M) Quantification of SAHF-positive cells. Average of three independent experiments (n=55-100 cells each) ± SEM. (C, E, G, and L) DNA was stained with DAPI (blue). (A, C, E, G, L) Scale bars represent 2 μm. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, Student’s unpaired t test. See also Figure S2 . Source data are provided as a Source Data file.

    Journal: bioRxiv

    Article Title: Persistent telomere cohesion protects aged cells from premature senescence

    doi: 10.1101/2020.02.03.932145

    Figure Lengend Snippet: Subtelomere recombination leads to DNA damage and premature senescence in aged human fibroblasts. (A) Immunofluorescence analysis of Vector, TRF1.WT, or TRF1.AA infected late (PD51) WI38 cells with RAD51 antibody. (B) Quantification of the frequency of cells displaying > 5 RAD51 foci. Average of two independent experiments (n=100 cells each) ± SEM. (C) Immunofluorescence analysis of TRF1.WT-infected late (PD51) WI38 cells with Rad51 (green) and TRF2 (red) antibodies. (D) Quantification of the frequency of cells displaying ≥2 Rad51/TRF2 colocalizations. Average of two independent experiments (n=100 cells each) ± SEM. (E) Immunofluorescence analysis of Vector, TRF1.WT, or TRF1.AA infected late (PD51) WI38 cells with γH2AX (green) and 53BP1 (red) antibodies. (F) Quantification of the frequency of cells displaying > 5 γH2AX/53BP1 colocalizing foci. Average of three independent experiments (n=55-100 cells each) ± SEM. (G) Immunofluorescence analysis of TRF1.WT-infected late (PD51) WI38 cells with γH2AX (green) and TIN2 (red) antibodies. (H) Quantification of the frequency of cells with ≥4 γH2AX/TIN2 colocalizing foci. Average of two independent experiments (n=100 cells each) ± SEM. (I) Growth curve analysis of Vector, TRF1.WT, or TRF1.AA infected late (PD52) WI38 cells. Average of two independent experiments ± SEM. (J) SA-β-gal analysis of Vector, TRF1.WT, or TRF1.AA infected late (PD51) WI38 cells. Scale bar represent 100 μm. (K) Quantification of SA-β-gal positive cells. Average of two independent experiments (n=50-100 cells each) ± SEM. (L) Detection of senescence-associated heterochromatin foci (SAHF) in Vector, TRF1.WT, or TRF1.AA infected late (PD51) WI38. (M) Quantification of SAHF-positive cells. Average of three independent experiments (n=55-100 cells each) ± SEM. (C, E, G, and L) DNA was stained with DAPI (blue). (A, C, E, G, L) Scale bars represent 2 μm. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, Student’s unpaired t test. See also Figure S2 . Source data are provided as a Source Data file.

    Article Snippet: SV40 LT immortalization of WI38 cells For SV40 Large T antigen infection, amphotropic retroviruses were generated by transfecting 20 µg of pBabe-neoLargeTcDNA into Phoenix Amphotropic cells (ATCC) using calcium phosphate precipitation.

    Techniques: Immunofluorescence, Plasmid Preparation, Infection, Staining

    Loss of the DNA damage checkpoint rescues TRF1-induced premature senescence and leads to runaway subtelomere recombination. (A) Growth curve analysis of Vector or SV40-LT infected mid (PD42) WI38 cells. Subsequent analyses were performed at PD6 (indicated) when Vector-infected cells begin to senesce. (B) FISH analysis of Vector or SV40-LT infected WI38 mitotic cells at PD6 using a 16p telo probe (green). (C and D) Quantification of the frequency of mitotic cells (C) with cohered telomeres or (D) exhibiting subtelomere copying in Vector or SV40-LT-infected WI38 cells at PD6. Average of two independent experiments (n=41-60 cells each) ± SEM. (E) Immunoblot analysis of Vector, TRF1.WT, or TRF1.AA infected WI38 SV40-LT (PD6) cell extracts. (F) FISH analysis of Vector, TRF1.WT, or TRF1.AA infected WI38 SV40-LT (PD6) cells on Day 1 of growth curve analysis (H) using a 16p telo probe (green). (G) Quantification of the frequency of mitotic cells with cohered telomeres. Average of two independent experiments (n=43-50 cells each) ± SEM. (H) Growth curves of Vector, TRF1.WT, or TRF1.AA WI38 infected SV40-LT (PD6) cells. Average of two independent experiments ± SEM. (I) FISH analysis of TRF1.WT infected WI38 SV40-LT (PD6) mitotic cells exhibiting subtelomere copying on Day 1 and Day 4 of growth curve analysis (F) using a 16p telo probe (green). (J) Quantification of the frequency of mitotic cells exhibiting subtelomere copying. Average of two independent experiments (n=40-50 cells each) ± SEM. (B, G, I) DNA was stained with DAPI (blue). Scale bars represent 2 μm. *p ≤ 0.05, **p ≤ 0.01, Student’s unpaired t test. See also Figure S3 . Source data are provided as a Source Data file.

    Journal: bioRxiv

    Article Title: Persistent telomere cohesion protects aged cells from premature senescence

    doi: 10.1101/2020.02.03.932145

    Figure Lengend Snippet: Loss of the DNA damage checkpoint rescues TRF1-induced premature senescence and leads to runaway subtelomere recombination. (A) Growth curve analysis of Vector or SV40-LT infected mid (PD42) WI38 cells. Subsequent analyses were performed at PD6 (indicated) when Vector-infected cells begin to senesce. (B) FISH analysis of Vector or SV40-LT infected WI38 mitotic cells at PD6 using a 16p telo probe (green). (C and D) Quantification of the frequency of mitotic cells (C) with cohered telomeres or (D) exhibiting subtelomere copying in Vector or SV40-LT-infected WI38 cells at PD6. Average of two independent experiments (n=41-60 cells each) ± SEM. (E) Immunoblot analysis of Vector, TRF1.WT, or TRF1.AA infected WI38 SV40-LT (PD6) cell extracts. (F) FISH analysis of Vector, TRF1.WT, or TRF1.AA infected WI38 SV40-LT (PD6) cells on Day 1 of growth curve analysis (H) using a 16p telo probe (green). (G) Quantification of the frequency of mitotic cells with cohered telomeres. Average of two independent experiments (n=43-50 cells each) ± SEM. (H) Growth curves of Vector, TRF1.WT, or TRF1.AA WI38 infected SV40-LT (PD6) cells. Average of two independent experiments ± SEM. (I) FISH analysis of TRF1.WT infected WI38 SV40-LT (PD6) mitotic cells exhibiting subtelomere copying on Day 1 and Day 4 of growth curve analysis (F) using a 16p telo probe (green). (J) Quantification of the frequency of mitotic cells exhibiting subtelomere copying. Average of two independent experiments (n=40-50 cells each) ± SEM. (B, G, I) DNA was stained with DAPI (blue). Scale bars represent 2 μm. *p ≤ 0.05, **p ≤ 0.01, Student’s unpaired t test. See also Figure S3 . Source data are provided as a Source Data file.

    Article Snippet: SV40 LT immortalization of WI38 cells For SV40 Large T antigen infection, amphotropic retroviruses were generated by transfecting 20 µg of pBabe-neoLargeTcDNA into Phoenix Amphotropic cells (ATCC) using calcium phosphate precipitation.

    Techniques: Plasmid Preparation, Infection, Fluorescence In Situ Hybridization, Staining

    TRF1-mediated recruitment of tankyrase 1 controls resolution of cohesion and suppresses Rad51-dependent subtelomere recombination in aged human fibroblasts. (A) FISH analysis of HEK293T wild-type (#23) and TRF1.G18P mutant (#1, #3, #5) mitotic cells with a 16p (triploid in HEK293T cells) telo probe (green). (B) Quantification of the frequency of mitotic cells with cohered telomeres. Average of three independent experiments (n=28-69 cells each) ± SEM. (C) FISH analysis of early (PD30) and late (PD54) passage WI38 mitotic cells with a 16p telo probe (green). (D) Quantification of the frequency of mitotic cells with cohered telomeres. Average of two independent experiments (n=36-50 cells each) ± SEM. (E) Immunoblot analysis of Vector, TRF1.WT, or TRF1.AA transfected late (PD52) WI38 cell extracts. (F) Immunofluorescence analysis of Vector, TRF1.WT, or TRF1.AA transfected late (PD52) WI38 cells using Myc (red) and TNKS1 (green) antibodies. (G) FISH analysis of Vector, TRF1.WT, or TRF1.AA transfected late (PD52) WI38 mitotic cells using a 16p telo probe (green). (H) Quantification of the frequency of mitotic cells with cohered telomeres. Average of two independent experiments (n=33-50 cells each) ± SEM. (I) FISH analysis of a TRF1.WT transfected late (PD52) WI38 mitotic cell exhibiting subtelomere copying (arrowhead) using a 16p telo probe (green). (J) Quantification of the frequency of mitotic cells exhibiting subtelomere copying. Average of two independent experiments (n=33-50 cells each) ± SEM. (A, C, F, G, I) DNA was stained with DAPI (blue). Scale bars represent 2 μm. *p ≤ 0.05, **p ≤ 0.01, ****p ≤ 0.0001, Student’s unpaired t test. See also Figure S1 . Source data are provided as a Source Data file.

    Journal: bioRxiv

    Article Title: Persistent telomere cohesion protects aged cells from premature senescence

    doi: 10.1101/2020.02.03.932145

    Figure Lengend Snippet: TRF1-mediated recruitment of tankyrase 1 controls resolution of cohesion and suppresses Rad51-dependent subtelomere recombination in aged human fibroblasts. (A) FISH analysis of HEK293T wild-type (#23) and TRF1.G18P mutant (#1, #3, #5) mitotic cells with a 16p (triploid in HEK293T cells) telo probe (green). (B) Quantification of the frequency of mitotic cells with cohered telomeres. Average of three independent experiments (n=28-69 cells each) ± SEM. (C) FISH analysis of early (PD30) and late (PD54) passage WI38 mitotic cells with a 16p telo probe (green). (D) Quantification of the frequency of mitotic cells with cohered telomeres. Average of two independent experiments (n=36-50 cells each) ± SEM. (E) Immunoblot analysis of Vector, TRF1.WT, or TRF1.AA transfected late (PD52) WI38 cell extracts. (F) Immunofluorescence analysis of Vector, TRF1.WT, or TRF1.AA transfected late (PD52) WI38 cells using Myc (red) and TNKS1 (green) antibodies. (G) FISH analysis of Vector, TRF1.WT, or TRF1.AA transfected late (PD52) WI38 mitotic cells using a 16p telo probe (green). (H) Quantification of the frequency of mitotic cells with cohered telomeres. Average of two independent experiments (n=33-50 cells each) ± SEM. (I) FISH analysis of a TRF1.WT transfected late (PD52) WI38 mitotic cell exhibiting subtelomere copying (arrowhead) using a 16p telo probe (green). (J) Quantification of the frequency of mitotic cells exhibiting subtelomere copying. Average of two independent experiments (n=33-50 cells each) ± SEM. (A, C, F, G, I) DNA was stained with DAPI (blue). Scale bars represent 2 μm. *p ≤ 0.05, **p ≤ 0.01, ****p ≤ 0.0001, Student’s unpaired t test. See also Figure S1 . Source data are provided as a Source Data file.

    Article Snippet: SV40 LT immortalization of WI38 cells For SV40 Large T antigen infection, amphotropic retroviruses were generated by transfecting 20 µg of pBabe-neoLargeTcDNA into Phoenix Amphotropic cells (ATCC) using calcium phosphate precipitation.

    Techniques: Fluorescence In Situ Hybridization, Mutagenesis, Plasmid Preparation, Transfection, Immunofluorescence, Staining