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primary hs68 human foreskin fibroblasts  (ATCC)


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    ATCC primary hs68 human foreskin fibroblasts
    FIGURE 3 | hTERT variants regulate the TRF2:TRF1 ratio by transcriptional and posttranslational mechanisms. (A) TRF2 mRNA levels deter- mined by RT-PCR in young and senescent BJ, <t>Hs68,</t> and Wi38 fibroblast strains. (B) Western blots of young (Y) and senescent (S) fibroblast strains probed with the indicated antibodies. (C) TRF1 mRNA levels in young and senescent BJ cells and BJs expressing WT-hTERT, R865C-hTERT, and V144M-hTERT variants. (D) Western blots of young and senescent BJ <t>fibroblasts,</t> and BJ cells expressing hTERT variants, probed with the indicated antibodies. (E) TRF2 protein levels in young and senescent fibroblast strains. (F) TRF2 protein levels in young and senescent BJs and BJs expressing hTERT variants. (A,C,E,F) Error bars = 95% CI; n = 3. p values determined by two-tailed unpaired t-tests with Welch's correction, ns = not significant. (G) Quantitation of the levels of the CDC20 and (H) FBXO5 Siah1 e3 ubiquitin ligases in cells expressing hTERT variants. Graphs show three bio- logical replicates with tables showing densitometry values for each replicate, with young BJ fibroblasts set to a value of 1. Error bars = 95% CI; n = 3. p values determined by two-tailed unpaired t-tests with Welch's correction. (I) Mock-transfected cells or cells expressing Siah1, CDC20, or FBXO5 were harvested 48 h after transfection, and lysates were blotted for the indicated proteins. GAPDH served as the loading control.
    Primary Hs68 Human Foreskin Fibroblasts, supplied by ATCC, used in various techniques. Bioz Stars score: 95/100, based on 136 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/primary+hs68+human+foreskin+fibroblasts/Hs68%3B+Foreskin+Fibroblast%3B+Human/pm40371663-209-0-5
    Average 95 stars, based on 136 article reviews
    primary hs68 human foreskin fibroblasts - by Bioz Stars, 2026-10
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    Images

    1) Product Images from "hTERT Increases TRF2 to Induce Telomere Compaction and Extend Cell Replicative Lifespan."

    Article Title: hTERT Increases TRF2 to Induce Telomere Compaction and Extend Cell Replicative Lifespan.

    Journal: Aging cell

    doi: 10.1111/acel.70105

    FIGURE 3 | hTERT variants regulate the TRF2:TRF1 ratio by transcriptional and posttranslational mechanisms. (A) TRF2 mRNA levels deter- mined by RT-PCR in young and senescent BJ, Hs68, and Wi38 fibroblast strains. (B) Western blots of young (Y) and senescent (S) fibroblast strains probed with the indicated antibodies. (C) TRF1 mRNA levels in young and senescent BJ cells and BJs expressing WT-hTERT, R865C-hTERT, and V144M-hTERT variants. (D) Western blots of young and senescent BJ fibroblasts, and BJ cells expressing hTERT variants, probed with the indicated antibodies. (E) TRF2 protein levels in young and senescent fibroblast strains. (F) TRF2 protein levels in young and senescent BJs and BJs expressing hTERT variants. (A,C,E,F) Error bars = 95% CI; n = 3. p values determined by two-tailed unpaired t-tests with Welch's correction, ns = not significant. (G) Quantitation of the levels of the CDC20 and (H) FBXO5 Siah1 e3 ubiquitin ligases in cells expressing hTERT variants. Graphs show three bio- logical replicates with tables showing densitometry values for each replicate, with young BJ fibroblasts set to a value of 1. Error bars = 95% CI; n = 3. p values determined by two-tailed unpaired t-tests with Welch's correction. (I) Mock-transfected cells or cells expressing Siah1, CDC20, or FBXO5 were harvested 48 h after transfection, and lysates were blotted for the indicated proteins. GAPDH served as the loading control.
    Figure Legend Snippet: FIGURE 3 | hTERT variants regulate the TRF2:TRF1 ratio by transcriptional and posttranslational mechanisms. (A) TRF2 mRNA levels deter- mined by RT-PCR in young and senescent BJ, Hs68, and Wi38 fibroblast strains. (B) Western blots of young (Y) and senescent (S) fibroblast strains probed with the indicated antibodies. (C) TRF1 mRNA levels in young and senescent BJ cells and BJs expressing WT-hTERT, R865C-hTERT, and V144M-hTERT variants. (D) Western blots of young and senescent BJ fibroblasts, and BJ cells expressing hTERT variants, probed with the indicated antibodies. (E) TRF2 protein levels in young and senescent fibroblast strains. (F) TRF2 protein levels in young and senescent BJs and BJs expressing hTERT variants. (A,C,E,F) Error bars = 95% CI; n = 3. p values determined by two-tailed unpaired t-tests with Welch's correction, ns = not significant. (G) Quantitation of the levels of the CDC20 and (H) FBXO5 Siah1 e3 ubiquitin ligases in cells expressing hTERT variants. Graphs show three bio- logical replicates with tables showing densitometry values for each replicate, with young BJ fibroblasts set to a value of 1. Error bars = 95% CI; n = 3. p values determined by two-tailed unpaired t-tests with Welch's correction. (I) Mock-transfected cells or cells expressing Siah1, CDC20, or FBXO5 were harvested 48 h after transfection, and lysates were blotted for the indicated proteins. GAPDH served as the loading control.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Two Tailed Test, Quantitation Assay, Ubiquitin Proteomics, Transfection, Control

    FIGURE 4 | Super-resolution imaging indicates that both long and dysfunctional telomeres cluster in interphase. (A) Representative structured illumination microscopy (SIM) immunofluorescence images of interphase BJ cells stained with PNA-TelC488 probes (green), 53BP1 (magenta), merged telomeres with 53BP1, and corresponding metaphase spreads (DAPI = blue and telomeres = green). White arrows indicate telomere-free ends (scale bar = 5 μm). (B) Representative wide-field immunofluorescence images of telomere (magenta) staining in young (Y) and senescent (S) Hs68 cells in interphase (scale bar = 5 μm). (C) Quantification of telomere count per cell. Error bars = 95% CI; n = 3; p values determined by two-tailed t-tests with Welch's correction. (D) Metaphase spreads of Hs68 young (Y) and senescent (S) cells. Blue = DAPI and green = telomere staining. White crosses indicate telomere-free ends (scale bar = 5 μm). (E) Quantification of the number of resolvable telomeres per cell based on interphase and metaphase cell staining in Panel D.
    Figure Legend Snippet: FIGURE 4 | Super-resolution imaging indicates that both long and dysfunctional telomeres cluster in interphase. (A) Representative structured illumination microscopy (SIM) immunofluorescence images of interphase BJ cells stained with PNA-TelC488 probes (green), 53BP1 (magenta), merged telomeres with 53BP1, and corresponding metaphase spreads (DAPI = blue and telomeres = green). White arrows indicate telomere-free ends (scale bar = 5 μm). (B) Representative wide-field immunofluorescence images of telomere (magenta) staining in young (Y) and senescent (S) Hs68 cells in interphase (scale bar = 5 μm). (C) Quantification of telomere count per cell. Error bars = 95% CI; n = 3; p values determined by two-tailed t-tests with Welch's correction. (D) Metaphase spreads of Hs68 young (Y) and senescent (S) cells. Blue = DAPI and green = telomere staining. White crosses indicate telomere-free ends (scale bar = 5 μm). (E) Quantification of the number of resolvable telomeres per cell based on interphase and metaphase cell staining in Panel D.

    Techniques Used: Imaging, Microscopy, Immunofluorescence, Staining, Two Tailed Test

    FIGURE 5 | Multiple telomeres cluster in 53BP1-foci in senescent cells. (A) Representative confocal immunofluorescence images of lamin A/C (magenta), 53BP1 (red), and telomere (green) staining in young (Y) and senescent (S) Hs68 cells (scale bars = 5 μm). (B) Quantification of the total amount of 53BP1 foci per cell. Error bars = 95% CI; n = 3 with > 50 cells counted per biological replicate. p values were determined by two-tailed t-tests with Welch's correction. (C) Quantification of the colocalization of 53BP1 with telomeres in young and senescent Hs68 cells. Over 50 cells were analyzed for young and senescent samples. (D) Airyscan image of a senescent Hs68 cell stained with PNA-TelC488 probes (green) and 53BP1 (red) (scale bar = 5 μm). (E) Inset enlargement of the boxed area in (D shows clustering of two and three telomeres within each 53BP1 focus. Quantification of the numbers of telomeres that colocalize with 53BP1 foci in (F) young and (G) senescent BJ and (H) young and (I) senescent Hs68 fibroblasts. A total of 55–100 foci were examined for each cell type and age. (J) Pooled data for BJ and Hs68 fibroblasts showed that the increase in colocalization of 53BP1 with telomeres in senescent cells occurred in both strains and was significant with Pearson's c2 = 40.63 and p = 1.84e-10.
    Figure Legend Snippet: FIGURE 5 | Multiple telomeres cluster in 53BP1-foci in senescent cells. (A) Representative confocal immunofluorescence images of lamin A/C (magenta), 53BP1 (red), and telomere (green) staining in young (Y) and senescent (S) Hs68 cells (scale bars = 5 μm). (B) Quantification of the total amount of 53BP1 foci per cell. Error bars = 95% CI; n = 3 with > 50 cells counted per biological replicate. p values were determined by two-tailed t-tests with Welch's correction. (C) Quantification of the colocalization of 53BP1 with telomeres in young and senescent Hs68 cells. Over 50 cells were analyzed for young and senescent samples. (D) Airyscan image of a senescent Hs68 cell stained with PNA-TelC488 probes (green) and 53BP1 (red) (scale bar = 5 μm). (E) Inset enlargement of the boxed area in (D shows clustering of two and three telomeres within each 53BP1 focus. Quantification of the numbers of telomeres that colocalize with 53BP1 foci in (F) young and (G) senescent BJ and (H) young and (I) senescent Hs68 fibroblasts. A total of 55–100 foci were examined for each cell type and age. (J) Pooled data for BJ and Hs68 fibroblasts showed that the increase in colocalization of 53BP1 with telomeres in senescent cells occurred in both strains and was significant with Pearson's c2 = 40.63 and p = 1.84e-10.

    Techniques Used: Immunofluorescence, Staining, Two Tailed Test

    Related Articles

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: hTERT Increases TRF2 to Induce Telomere Compaction and Extend Cell Replicative Lifespan
    Article Snippet: Primary Hs68 human foreskin fibroblasts (ATCC CRL‐1635) and WI38 fetal lung fibroblasts (ATCC CCL‐75) were grown in DMEM, 1 g/L glucose (Gibco, 11885‐084), supplemented with 10% (v/v) FBS.

    Article Title: hTERT Increases TRF2 to Induce Telomere Compaction and Extend Cell Replicative Lifespan.
    Article Snippet: Primary Hs68 human foreskin fibroblasts (ATCC CRL- 1635) and WI38 fetal lung fibroblasts (ATCC CCL- 75) were grown in DMEM, 1 g/L glucose (Gibco, 11885- 084), supplemented with 10% (v/v) FBS.

    Western Blot:

    Article Title: hTERT Increases TRF2 to Induce Telomere Compaction and Extend Cell Replicative Lifespan
    Article Snippet: Primary Hs68 human foreskin fibroblasts (ATCC CRL‐1635) and WI38 fetal lung fibroblasts (ATCC CCL‐75) were grown in DMEM, 1 g/L glucose (Gibco, 11885‐084), supplemented with 10% (v/v) FBS.

    Article Title: hTERT Increases TRF2 to Induce Telomere Compaction and Extend Cell Replicative Lifespan.
    Article Snippet: Primary Hs68 human foreskin fibroblasts (ATCC CRL- 1635) and WI38 fetal lung fibroblasts (ATCC CCL- 75) were grown in DMEM, 1 g/L glucose (Gibco, 11885- 084), supplemented with 10% (v/v) FBS.

    Expressing:

    Article Title: hTERT Increases TRF2 to Induce Telomere Compaction and Extend Cell Replicative Lifespan
    Article Snippet: Primary Hs68 human foreskin fibroblasts (ATCC CRL‐1635) and WI38 fetal lung fibroblasts (ATCC CCL‐75) were grown in DMEM, 1 g/L glucose (Gibco, 11885‐084), supplemented with 10% (v/v) FBS.

    Article Title: hTERT Increases TRF2 to Induce Telomere Compaction and Extend Cell Replicative Lifespan.
    Article Snippet: Primary Hs68 human foreskin fibroblasts (ATCC CRL- 1635) and WI38 fetal lung fibroblasts (ATCC CCL- 75) were grown in DMEM, 1 g/L glucose (Gibco, 11885- 084), supplemented with 10% (v/v) FBS.

    Two Tailed Test:

    Article Title: hTERT Increases TRF2 to Induce Telomere Compaction and Extend Cell Replicative Lifespan
    Article Snippet: Primary Hs68 human foreskin fibroblasts (ATCC CRL‐1635) and WI38 fetal lung fibroblasts (ATCC CCL‐75) were grown in DMEM, 1 g/L glucose (Gibco, 11885‐084), supplemented with 10% (v/v) FBS.

    Article Title: hTERT Increases TRF2 to Induce Telomere Compaction and Extend Cell Replicative Lifespan.
    Article Snippet: Primary Hs68 human foreskin fibroblasts (ATCC CRL- 1635) and WI38 fetal lung fibroblasts (ATCC CCL- 75) were grown in DMEM, 1 g/L glucose (Gibco, 11885- 084), supplemented with 10% (v/v) FBS.

    Quantitation Assay:

    Article Title: hTERT Increases TRF2 to Induce Telomere Compaction and Extend Cell Replicative Lifespan
    Article Snippet: Primary Hs68 human foreskin fibroblasts (ATCC CRL‐1635) and WI38 fetal lung fibroblasts (ATCC CCL‐75) were grown in DMEM, 1 g/L glucose (Gibco, 11885‐084), supplemented with 10% (v/v) FBS.

    Article Title: hTERT Increases TRF2 to Induce Telomere Compaction and Extend Cell Replicative Lifespan.
    Article Snippet: Primary Hs68 human foreskin fibroblasts (ATCC CRL- 1635) and WI38 fetal lung fibroblasts (ATCC CCL- 75) were grown in DMEM, 1 g/L glucose (Gibco, 11885- 084), supplemented with 10% (v/v) FBS.

    Ubiquitin Proteomics:

    Article Title: hTERT Increases TRF2 to Induce Telomere Compaction and Extend Cell Replicative Lifespan
    Article Snippet: Primary Hs68 human foreskin fibroblasts (ATCC CRL‐1635) and WI38 fetal lung fibroblasts (ATCC CCL‐75) were grown in DMEM, 1 g/L glucose (Gibco, 11885‐084), supplemented with 10% (v/v) FBS.

    Article Title: hTERT Increases TRF2 to Induce Telomere Compaction and Extend Cell Replicative Lifespan.
    Article Snippet: Primary Hs68 human foreskin fibroblasts (ATCC CRL- 1635) and WI38 fetal lung fibroblasts (ATCC CCL- 75) were grown in DMEM, 1 g/L glucose (Gibco, 11885- 084), supplemented with 10% (v/v) FBS.

    Transfection:

    Article Title: hTERT Increases TRF2 to Induce Telomere Compaction and Extend Cell Replicative Lifespan
    Article Snippet: Primary Hs68 human foreskin fibroblasts (ATCC CRL‐1635) and WI38 fetal lung fibroblasts (ATCC CCL‐75) were grown in DMEM, 1 g/L glucose (Gibco, 11885‐084), supplemented with 10% (v/v) FBS.

    Article Title: hTERT Increases TRF2 to Induce Telomere Compaction and Extend Cell Replicative Lifespan.
    Article Snippet: Primary Hs68 human foreskin fibroblasts (ATCC CRL- 1635) and WI38 fetal lung fibroblasts (ATCC CCL- 75) were grown in DMEM, 1 g/L glucose (Gibco, 11885- 084), supplemented with 10% (v/v) FBS.

    Control:

    Article Title: hTERT Increases TRF2 to Induce Telomere Compaction and Extend Cell Replicative Lifespan
    Article Snippet: Primary Hs68 human foreskin fibroblasts (ATCC CRL‐1635) and WI38 fetal lung fibroblasts (ATCC CCL‐75) were grown in DMEM, 1 g/L glucose (Gibco, 11885‐084), supplemented with 10% (v/v) FBS.

    Article Title: hTERT Increases TRF2 to Induce Telomere Compaction and Extend Cell Replicative Lifespan.
    Article Snippet: Primary Hs68 human foreskin fibroblasts (ATCC CRL- 1635) and WI38 fetal lung fibroblasts (ATCC CCL- 75) were grown in DMEM, 1 g/L glucose (Gibco, 11885- 084), supplemented with 10% (v/v) FBS.

    Imaging:

    Article Title: hTERT Increases TRF2 to Induce Telomere Compaction and Extend Cell Replicative Lifespan
    Article Snippet: Primary Hs68 human foreskin fibroblasts (ATCC CRL‐1635) and WI38 fetal lung fibroblasts (ATCC CCL‐75) were grown in DMEM, 1 g/L glucose (Gibco, 11885‐084), supplemented with 10% (v/v) FBS.

    Article Title: hTERT Increases TRF2 to Induce Telomere Compaction and Extend Cell Replicative Lifespan.
    Article Snippet: Primary Hs68 human foreskin fibroblasts (ATCC CRL- 1635) and WI38 fetal lung fibroblasts (ATCC CCL- 75) were grown in DMEM, 1 g/L glucose (Gibco, 11885- 084), supplemented with 10% (v/v) FBS.

    Microscopy:

    Article Title: hTERT Increases TRF2 to Induce Telomere Compaction and Extend Cell Replicative Lifespan
    Article Snippet: Primary Hs68 human foreskin fibroblasts (ATCC CRL‐1635) and WI38 fetal lung fibroblasts (ATCC CCL‐75) were grown in DMEM, 1 g/L glucose (Gibco, 11885‐084), supplemented with 10% (v/v) FBS.

    Article Title: hTERT Increases TRF2 to Induce Telomere Compaction and Extend Cell Replicative Lifespan.
    Article Snippet: Primary Hs68 human foreskin fibroblasts (ATCC CRL- 1635) and WI38 fetal lung fibroblasts (ATCC CCL- 75) were grown in DMEM, 1 g/L glucose (Gibco, 11885- 084), supplemented with 10% (v/v) FBS.

    Immunofluorescence:

    Article Title: hTERT Increases TRF2 to Induce Telomere Compaction and Extend Cell Replicative Lifespan
    Article Snippet: Primary Hs68 human foreskin fibroblasts (ATCC CRL‐1635) and WI38 fetal lung fibroblasts (ATCC CCL‐75) were grown in DMEM, 1 g/L glucose (Gibco, 11885‐084), supplemented with 10% (v/v) FBS.

    Article Title: hTERT Increases TRF2 to Induce Telomere Compaction and Extend Cell Replicative Lifespan.
    Article Snippet: Primary Hs68 human foreskin fibroblasts (ATCC CRL- 1635) and WI38 fetal lung fibroblasts (ATCC CCL- 75) were grown in DMEM, 1 g/L glucose (Gibco, 11885- 084), supplemented with 10% (v/v) FBS.

    Staining:

    Article Title: hTERT Increases TRF2 to Induce Telomere Compaction and Extend Cell Replicative Lifespan
    Article Snippet: Primary Hs68 human foreskin fibroblasts (ATCC CRL‐1635) and WI38 fetal lung fibroblasts (ATCC CCL‐75) were grown in DMEM, 1 g/L glucose (Gibco, 11885‐084), supplemented with 10% (v/v) FBS.

    Article Title: hTERT Increases TRF2 to Induce Telomere Compaction and Extend Cell Replicative Lifespan.
    Article Snippet: Primary Hs68 human foreskin fibroblasts (ATCC CRL- 1635) and WI38 fetal lung fibroblasts (ATCC CCL- 75) were grown in DMEM, 1 g/L glucose (Gibco, 11885- 084), supplemented with 10% (v/v) FBS.



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    FIGURE 3 | hTERT variants regulate the TRF2:TRF1 ratio by transcriptional and posttranslational mechanisms. (A) TRF2 mRNA levels deter- mined by RT-PCR in young and senescent BJ, <t>Hs68,</t> and Wi38 fibroblast strains. (B) Western blots of young (Y) and senescent (S) fibroblast strains probed with the indicated antibodies. (C) TRF1 mRNA levels in young and senescent BJ cells and BJs expressing WT-hTERT, R865C-hTERT, and V144M-hTERT variants. (D) Western blots of young and senescent BJ <t>fibroblasts,</t> and BJ cells expressing hTERT variants, probed with the indicated antibodies. (E) TRF2 protein levels in young and senescent fibroblast strains. (F) TRF2 protein levels in young and senescent BJs and BJs expressing hTERT variants. (A,C,E,F) Error bars = 95% CI; n = 3. p values determined by two-tailed unpaired t-tests with Welch's correction, ns = not significant. (G) Quantitation of the levels of the CDC20 and (H) FBXO5 Siah1 e3 ubiquitin ligases in cells expressing hTERT variants. Graphs show three bio- logical replicates with tables showing densitometry values for each replicate, with young BJ fibroblasts set to a value of 1. Error bars = 95% CI; n = 3. p values determined by two-tailed unpaired t-tests with Welch's correction. (I) Mock-transfected cells or cells expressing Siah1, CDC20, or FBXO5 were harvested 48 h after transfection, and lysates were blotted for the indicated proteins. GAPDH served as the loading control.
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    Image Search Results


    FIGURE 3 | hTERT variants regulate the TRF2:TRF1 ratio by transcriptional and posttranslational mechanisms. (A) TRF2 mRNA levels deter- mined by RT-PCR in young and senescent BJ, Hs68, and Wi38 fibroblast strains. (B) Western blots of young (Y) and senescent (S) fibroblast strains probed with the indicated antibodies. (C) TRF1 mRNA levels in young and senescent BJ cells and BJs expressing WT-hTERT, R865C-hTERT, and V144M-hTERT variants. (D) Western blots of young and senescent BJ fibroblasts, and BJ cells expressing hTERT variants, probed with the indicated antibodies. (E) TRF2 protein levels in young and senescent fibroblast strains. (F) TRF2 protein levels in young and senescent BJs and BJs expressing hTERT variants. (A,C,E,F) Error bars = 95% CI; n = 3. p values determined by two-tailed unpaired t-tests with Welch's correction, ns = not significant. (G) Quantitation of the levels of the CDC20 and (H) FBXO5 Siah1 e3 ubiquitin ligases in cells expressing hTERT variants. Graphs show three bio- logical replicates with tables showing densitometry values for each replicate, with young BJ fibroblasts set to a value of 1. Error bars = 95% CI; n = 3. p values determined by two-tailed unpaired t-tests with Welch's correction. (I) Mock-transfected cells or cells expressing Siah1, CDC20, or FBXO5 were harvested 48 h after transfection, and lysates were blotted for the indicated proteins. GAPDH served as the loading control.

    Journal: Aging cell

    Article Title: hTERT Increases TRF2 to Induce Telomere Compaction and Extend Cell Replicative Lifespan.

    doi: 10.1111/acel.70105

    Figure Lengend Snippet: FIGURE 3 | hTERT variants regulate the TRF2:TRF1 ratio by transcriptional and posttranslational mechanisms. (A) TRF2 mRNA levels deter- mined by RT-PCR in young and senescent BJ, Hs68, and Wi38 fibroblast strains. (B) Western blots of young (Y) and senescent (S) fibroblast strains probed with the indicated antibodies. (C) TRF1 mRNA levels in young and senescent BJ cells and BJs expressing WT-hTERT, R865C-hTERT, and V144M-hTERT variants. (D) Western blots of young and senescent BJ fibroblasts, and BJ cells expressing hTERT variants, probed with the indicated antibodies. (E) TRF2 protein levels in young and senescent fibroblast strains. (F) TRF2 protein levels in young and senescent BJs and BJs expressing hTERT variants. (A,C,E,F) Error bars = 95% CI; n = 3. p values determined by two-tailed unpaired t-tests with Welch's correction, ns = not significant. (G) Quantitation of the levels of the CDC20 and (H) FBXO5 Siah1 e3 ubiquitin ligases in cells expressing hTERT variants. Graphs show three bio- logical replicates with tables showing densitometry values for each replicate, with young BJ fibroblasts set to a value of 1. Error bars = 95% CI; n = 3. p values determined by two-tailed unpaired t-tests with Welch's correction. (I) Mock-transfected cells or cells expressing Siah1, CDC20, or FBXO5 were harvested 48 h after transfection, and lysates were blotted for the indicated proteins. GAPDH served as the loading control.

    Article Snippet: Primary Hs68 human foreskin fibroblasts (ATCC CRL- 1635) and WI38 fetal lung fibroblasts (ATCC CCL- 75) were grown in DMEM, 1 g/L glucose (Gibco, 11885- 084), supplemented with 10% (v/v) FBS.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Two Tailed Test, Quantitation Assay, Ubiquitin Proteomics, Transfection, Control

    FIGURE 4 | Super-resolution imaging indicates that both long and dysfunctional telomeres cluster in interphase. (A) Representative structured illumination microscopy (SIM) immunofluorescence images of interphase BJ cells stained with PNA-TelC488 probes (green), 53BP1 (magenta), merged telomeres with 53BP1, and corresponding metaphase spreads (DAPI = blue and telomeres = green). White arrows indicate telomere-free ends (scale bar = 5 μm). (B) Representative wide-field immunofluorescence images of telomere (magenta) staining in young (Y) and senescent (S) Hs68 cells in interphase (scale bar = 5 μm). (C) Quantification of telomere count per cell. Error bars = 95% CI; n = 3; p values determined by two-tailed t-tests with Welch's correction. (D) Metaphase spreads of Hs68 young (Y) and senescent (S) cells. Blue = DAPI and green = telomere staining. White crosses indicate telomere-free ends (scale bar = 5 μm). (E) Quantification of the number of resolvable telomeres per cell based on interphase and metaphase cell staining in Panel D.

    Journal: Aging cell

    Article Title: hTERT Increases TRF2 to Induce Telomere Compaction and Extend Cell Replicative Lifespan.

    doi: 10.1111/acel.70105

    Figure Lengend Snippet: FIGURE 4 | Super-resolution imaging indicates that both long and dysfunctional telomeres cluster in interphase. (A) Representative structured illumination microscopy (SIM) immunofluorescence images of interphase BJ cells stained with PNA-TelC488 probes (green), 53BP1 (magenta), merged telomeres with 53BP1, and corresponding metaphase spreads (DAPI = blue and telomeres = green). White arrows indicate telomere-free ends (scale bar = 5 μm). (B) Representative wide-field immunofluorescence images of telomere (magenta) staining in young (Y) and senescent (S) Hs68 cells in interphase (scale bar = 5 μm). (C) Quantification of telomere count per cell. Error bars = 95% CI; n = 3; p values determined by two-tailed t-tests with Welch's correction. (D) Metaphase spreads of Hs68 young (Y) and senescent (S) cells. Blue = DAPI and green = telomere staining. White crosses indicate telomere-free ends (scale bar = 5 μm). (E) Quantification of the number of resolvable telomeres per cell based on interphase and metaphase cell staining in Panel D.

    Article Snippet: Primary Hs68 human foreskin fibroblasts (ATCC CRL- 1635) and WI38 fetal lung fibroblasts (ATCC CCL- 75) were grown in DMEM, 1 g/L glucose (Gibco, 11885- 084), supplemented with 10% (v/v) FBS.

    Techniques: Imaging, Microscopy, Immunofluorescence, Staining, Two Tailed Test

    FIGURE 5 | Multiple telomeres cluster in 53BP1-foci in senescent cells. (A) Representative confocal immunofluorescence images of lamin A/C (magenta), 53BP1 (red), and telomere (green) staining in young (Y) and senescent (S) Hs68 cells (scale bars = 5 μm). (B) Quantification of the total amount of 53BP1 foci per cell. Error bars = 95% CI; n = 3 with > 50 cells counted per biological replicate. p values were determined by two-tailed t-tests with Welch's correction. (C) Quantification of the colocalization of 53BP1 with telomeres in young and senescent Hs68 cells. Over 50 cells were analyzed for young and senescent samples. (D) Airyscan image of a senescent Hs68 cell stained with PNA-TelC488 probes (green) and 53BP1 (red) (scale bar = 5 μm). (E) Inset enlargement of the boxed area in (D shows clustering of two and three telomeres within each 53BP1 focus. Quantification of the numbers of telomeres that colocalize with 53BP1 foci in (F) young and (G) senescent BJ and (H) young and (I) senescent Hs68 fibroblasts. A total of 55–100 foci were examined for each cell type and age. (J) Pooled data for BJ and Hs68 fibroblasts showed that the increase in colocalization of 53BP1 with telomeres in senescent cells occurred in both strains and was significant with Pearson's c2 = 40.63 and p = 1.84e-10.

    Journal: Aging cell

    Article Title: hTERT Increases TRF2 to Induce Telomere Compaction and Extend Cell Replicative Lifespan.

    doi: 10.1111/acel.70105

    Figure Lengend Snippet: FIGURE 5 | Multiple telomeres cluster in 53BP1-foci in senescent cells. (A) Representative confocal immunofluorescence images of lamin A/C (magenta), 53BP1 (red), and telomere (green) staining in young (Y) and senescent (S) Hs68 cells (scale bars = 5 μm). (B) Quantification of the total amount of 53BP1 foci per cell. Error bars = 95% CI; n = 3 with > 50 cells counted per biological replicate. p values were determined by two-tailed t-tests with Welch's correction. (C) Quantification of the colocalization of 53BP1 with telomeres in young and senescent Hs68 cells. Over 50 cells were analyzed for young and senescent samples. (D) Airyscan image of a senescent Hs68 cell stained with PNA-TelC488 probes (green) and 53BP1 (red) (scale bar = 5 μm). (E) Inset enlargement of the boxed area in (D shows clustering of two and three telomeres within each 53BP1 focus. Quantification of the numbers of telomeres that colocalize with 53BP1 foci in (F) young and (G) senescent BJ and (H) young and (I) senescent Hs68 fibroblasts. A total of 55–100 foci were examined for each cell type and age. (J) Pooled data for BJ and Hs68 fibroblasts showed that the increase in colocalization of 53BP1 with telomeres in senescent cells occurred in both strains and was significant with Pearson's c2 = 40.63 and p = 1.84e-10.

    Article Snippet: Primary Hs68 human foreskin fibroblasts (ATCC CRL- 1635) and WI38 fetal lung fibroblasts (ATCC CCL- 75) were grown in DMEM, 1 g/L glucose (Gibco, 11885- 084), supplemented with 10% (v/v) FBS.

    Techniques: Immunofluorescence, Staining, Two Tailed Test

    Fig. 2 Metaphase spreads of a, b Hs68 and c, d Hs68 + hTERT cells. DNA was stained with DAPI (blue) and telomeres were stained with PNA- TelC488-conjugated probe (green). a, c Chromosomes were manually counted and labelled in ImageJ. Both Hs68 and Hs68 + hTERT cells counted 46 chromosomes confirming a diploid genome. b, d Telomere staining indicated no telomere-free ends in both Hs68 and Hs68 + hTERT cells. Images were acquired using a Nikon Ti Eclipse Widefield system and deconvolved. Scale bar = 5 µm.

    Journal: Communications biology

    Article Title: Telomere analysis using 3D fluorescence microscopy suggests mammalian telomere clustering in hTERT-immortalized Hs68 fibroblasts.

    doi: 10.1038/s42003-019-0692-z

    Figure Lengend Snippet: Fig. 2 Metaphase spreads of a, b Hs68 and c, d Hs68 + hTERT cells. DNA was stained with DAPI (blue) and telomeres were stained with PNA- TelC488-conjugated probe (green). a, c Chromosomes were manually counted and labelled in ImageJ. Both Hs68 and Hs68 + hTERT cells counted 46 chromosomes confirming a diploid genome. b, d Telomere staining indicated no telomere-free ends in both Hs68 and Hs68 + hTERT cells. Images were acquired using a Nikon Ti Eclipse Widefield system and deconvolved. Scale bar = 5 µm.

    Article Snippet: Primary human foreskin fibroblasts Hs68 (CRL-1635, ATCC) cells were grown in DMEM, 1 g/L glucose (Gibco, 11885–084) containing 10% fetal bovine serum (FBS).

    Techniques: Staining

    Fig. 3 Correction of raw images using dark frame and flat-field images. a Raw images were acquired using the full field of view (FOV) of the camera. Hs68 cells were stained with DAPI (blue) and PNA-TelC647-conjugated probe (magenta). b Averaged dark frame image. c Flat-field image showing variable intensity across the field of view. d Plot profile intensity graph of the line drawn across the flat-field image in panel c. e Deconvolved images of the dark noise and flat-field corrected images of panel a. All images were taken using a Nikon Ti Eclipse Widefield system. Scale bar = 25 µm.

    Journal: Communications biology

    Article Title: Telomere analysis using 3D fluorescence microscopy suggests mammalian telomere clustering in hTERT-immortalized Hs68 fibroblasts.

    doi: 10.1038/s42003-019-0692-z

    Figure Lengend Snippet: Fig. 3 Correction of raw images using dark frame and flat-field images. a Raw images were acquired using the full field of view (FOV) of the camera. Hs68 cells were stained with DAPI (blue) and PNA-TelC647-conjugated probe (magenta). b Averaged dark frame image. c Flat-field image showing variable intensity across the field of view. d Plot profile intensity graph of the line drawn across the flat-field image in panel c. e Deconvolved images of the dark noise and flat-field corrected images of panel a. All images were taken using a Nikon Ti Eclipse Widefield system. Scale bar = 25 µm.

    Article Snippet: Primary human foreskin fibroblasts Hs68 (CRL-1635, ATCC) cells were grown in DMEM, 1 g/L glucose (Gibco, 11885–084) containing 10% fetal bovine serum (FBS).

    Techniques: Staining

    Fig. 4 2D analysis of telomeres in interphase Hs68 cells using the ImageJ program. a Example of an interphase Hs68 cell stained with PNA-TelC647- conjugated probe for telomeres (magenta) and DAPI (blue). Grayscale, deconvolved image of PNA TelC647 staining was used for telomere count using the Find Maxima program in ImageJ. b Graph showing average telomere counts per cell using different noise tolerances for Find Maxima (n = 3 biologically independent experiments, >50 cells per experiment). c Grayscale, raw image of PNA TelC647 staining and grayscale, deconvolved image of PNA TelC647 staining. Intensity-based thresholding (Otsu) of raw image and deconvolved image was performed. Red indicates the foreground signal that is separated from the background when applying the Otsu threshold. d Grayscale, deconvolved image of PNA TelC647 staining in an Hs68 cell. e Inset is an enlargement of the boxed area in panel d. f Plot profile along the purple arrow in the inset in panel e. The intensity graph indicates two spots, but they cannot be separated by thresholding and watershedding. All images were taken using a Nikon Ti Eclipse Widefield system. Scale bars = 5 µm. Source data used for the graphs in b can be found in Supplementary Data 1.

    Journal: Communications biology

    Article Title: Telomere analysis using 3D fluorescence microscopy suggests mammalian telomere clustering in hTERT-immortalized Hs68 fibroblasts.

    doi: 10.1038/s42003-019-0692-z

    Figure Lengend Snippet: Fig. 4 2D analysis of telomeres in interphase Hs68 cells using the ImageJ program. a Example of an interphase Hs68 cell stained with PNA-TelC647- conjugated probe for telomeres (magenta) and DAPI (blue). Grayscale, deconvolved image of PNA TelC647 staining was used for telomere count using the Find Maxima program in ImageJ. b Graph showing average telomere counts per cell using different noise tolerances for Find Maxima (n = 3 biologically independent experiments, >50 cells per experiment). c Grayscale, raw image of PNA TelC647 staining and grayscale, deconvolved image of PNA TelC647 staining. Intensity-based thresholding (Otsu) of raw image and deconvolved image was performed. Red indicates the foreground signal that is separated from the background when applying the Otsu threshold. d Grayscale, deconvolved image of PNA TelC647 staining in an Hs68 cell. e Inset is an enlargement of the boxed area in panel d. f Plot profile along the purple arrow in the inset in panel e. The intensity graph indicates two spots, but they cannot be separated by thresholding and watershedding. All images were taken using a Nikon Ti Eclipse Widefield system. Scale bars = 5 µm. Source data used for the graphs in b can be found in Supplementary Data 1.

    Article Snippet: Primary human foreskin fibroblasts Hs68 (CRL-1635, ATCC) cells were grown in DMEM, 1 g/L glucose (Gibco, 11885–084) containing 10% fetal bovine serum (FBS).

    Techniques: Staining

    Fig. 5 Analysing telomeres in 3D using Imaris. a Example of a deconvolved, 3D image of an Hs68 cell stained with PNA TelC647-conjugated probe (magenta) visualized in the Imaris program. b The centre of the fluorescence signal (small dots) was detected based on Gaussian filtering in Imaris. This was used to determine the location of each telomere within the image. c Visualization of the fluorescence signal of two telomeres (magenta) from above (XY) and from the side (Z). Spots were assigned to the fluorescence intensity that represents telomeric PNA probe. Overlay of the spots with the fluorescence intensity from above (XY) and from the side (Z) shows how accurate the assigned spots reflect telomeric signal. d Overlay of the spots with the fluorescence intensity representing the telomeres in this 3D image of an Hs68 cell. Spots were colour-coded based on volume and visualized from above. Scale bars = 2 µm.

    Journal: Communications biology

    Article Title: Telomere analysis using 3D fluorescence microscopy suggests mammalian telomere clustering in hTERT-immortalized Hs68 fibroblasts.

    doi: 10.1038/s42003-019-0692-z

    Figure Lengend Snippet: Fig. 5 Analysing telomeres in 3D using Imaris. a Example of a deconvolved, 3D image of an Hs68 cell stained with PNA TelC647-conjugated probe (magenta) visualized in the Imaris program. b The centre of the fluorescence signal (small dots) was detected based on Gaussian filtering in Imaris. This was used to determine the location of each telomere within the image. c Visualization of the fluorescence signal of two telomeres (magenta) from above (XY) and from the side (Z). Spots were assigned to the fluorescence intensity that represents telomeric PNA probe. Overlay of the spots with the fluorescence intensity from above (XY) and from the side (Z) shows how accurate the assigned spots reflect telomeric signal. d Overlay of the spots with the fluorescence intensity representing the telomeres in this 3D image of an Hs68 cell. Spots were colour-coded based on volume and visualized from above. Scale bars = 2 µm.

    Article Snippet: Primary human foreskin fibroblasts Hs68 (CRL-1635, ATCC) cells were grown in DMEM, 1 g/L glucose (Gibco, 11885–084) containing 10% fetal bovine serum (FBS).

    Techniques: Staining

    Fig. 6 Confirmation of hTERT expression and telomere elongation in the generated cell line Hs68 + hTERT. a Immunoprecipitation of Flag-hTERT in Hs68 and Hs68 + hTERT cells followed by western blot for hTERT protein. Low levels of hTERT were detected in input samples of Hs68, but no hTERT was detected after immunoprecipitation with Flag-antibody. In both input and immunoprecipitated samples of Hs68 + hTERT high levels of hTERT were detected, indicating successful infection with hTERT-plasmid. b RNA expression of hTERT in Hs68 and Hs68 + hTERT measured by qPCR. c Telomerase activity of hTERT in Hs68 and Hs68 + hTERT measured by Telomerase Repeated Amplification Protocol (TRAP). d Relative amount of telomeric DNA in Hs68 and Hs68 + hTERT measured by telo-qPCR. Upon hTERT overexpression, there is a 3.5-fold increase in average telomere length in Hs68 + hTERT compared to Hs68 cells. e Absolute telomere length in Hs68 (~5.3 kb) and Hs68 + hTERT (~18.9 kb) measured by Telomere Restriction Fragments (TRF) analysis. This matched the telo-qPCR data and a 3.5-fold increase in telomere length upon hTERT overexpression. Two-tailed unpaired t-tests were performed, n = 3 biologically independent experiments, ****P < 0.0001. Source data used for the graphs in b and d can be found in Supplementary Data 2.

    Journal: Communications biology

    Article Title: Telomere analysis using 3D fluorescence microscopy suggests mammalian telomere clustering in hTERT-immortalized Hs68 fibroblasts.

    doi: 10.1038/s42003-019-0692-z

    Figure Lengend Snippet: Fig. 6 Confirmation of hTERT expression and telomere elongation in the generated cell line Hs68 + hTERT. a Immunoprecipitation of Flag-hTERT in Hs68 and Hs68 + hTERT cells followed by western blot for hTERT protein. Low levels of hTERT were detected in input samples of Hs68, but no hTERT was detected after immunoprecipitation with Flag-antibody. In both input and immunoprecipitated samples of Hs68 + hTERT high levels of hTERT were detected, indicating successful infection with hTERT-plasmid. b RNA expression of hTERT in Hs68 and Hs68 + hTERT measured by qPCR. c Telomerase activity of hTERT in Hs68 and Hs68 + hTERT measured by Telomerase Repeated Amplification Protocol (TRAP). d Relative amount of telomeric DNA in Hs68 and Hs68 + hTERT measured by telo-qPCR. Upon hTERT overexpression, there is a 3.5-fold increase in average telomere length in Hs68 + hTERT compared to Hs68 cells. e Absolute telomere length in Hs68 (~5.3 kb) and Hs68 + hTERT (~18.9 kb) measured by Telomere Restriction Fragments (TRF) analysis. This matched the telo-qPCR data and a 3.5-fold increase in telomere length upon hTERT overexpression. Two-tailed unpaired t-tests were performed, n = 3 biologically independent experiments, ****P < 0.0001. Source data used for the graphs in b and d can be found in Supplementary Data 2.

    Article Snippet: Primary human foreskin fibroblasts Hs68 (CRL-1635, ATCC) cells were grown in DMEM, 1 g/L glucose (Gibco, 11885–084) containing 10% fetal bovine serum (FBS).

    Techniques: Expressing, Generated, Immunoprecipitation, Western Blot, Infection, Plasmid Preparation, RNA Expression, Activity Assay, Over Expression, Two Tailed Test

    Fig. 7 Hs68 + hTERT cells have longer and bigger telomeres when compared to Hs68. a Frequency distribution of the Intensity Sum of the individual spots assigned in Imaris in 3D. Intensity Sum represents telomere length of the telomeres in Hs68 and Hs68 + hTERT. b Histogram of the logarithm of the Intensity Sum of the spots. c Volume measurements of the individual spots in 3D. Telomeres in Hs68 + hTERT cells are on average twofold bigger when compared to telomeres in Hs68 cells. d, e The coefficient of determination, R2, demonstrates a non-linear correlation between intensity and volume of individual spots in Hs68 and Hs68 + hTERT. A weaker R2 is observed in Hs68 + hTERT cells compared to Hs68. Note the difference in X and Y axes when comparing Hs68 (d) with Hs68 + hTERT (e). f Telomere count in 2D using the Find Maxima setting in ImageJ and various noise tolerances. g Telomere count in 3D using Imaris ‘Spots’ feature. Hs68 + hTERT cells have on average less telomeres per cell compared to Hs68. Two-tailed unpaired t-tests were performed, n = 3 biologically independent experiments, ****P < 0.0001. Source data used for the graphs can be found in Supplementary Data 3.

    Journal: Communications biology

    Article Title: Telomere analysis using 3D fluorescence microscopy suggests mammalian telomere clustering in hTERT-immortalized Hs68 fibroblasts.

    doi: 10.1038/s42003-019-0692-z

    Figure Lengend Snippet: Fig. 7 Hs68 + hTERT cells have longer and bigger telomeres when compared to Hs68. a Frequency distribution of the Intensity Sum of the individual spots assigned in Imaris in 3D. Intensity Sum represents telomere length of the telomeres in Hs68 and Hs68 + hTERT. b Histogram of the logarithm of the Intensity Sum of the spots. c Volume measurements of the individual spots in 3D. Telomeres in Hs68 + hTERT cells are on average twofold bigger when compared to telomeres in Hs68 cells. d, e The coefficient of determination, R2, demonstrates a non-linear correlation between intensity and volume of individual spots in Hs68 and Hs68 + hTERT. A weaker R2 is observed in Hs68 + hTERT cells compared to Hs68. Note the difference in X and Y axes when comparing Hs68 (d) with Hs68 + hTERT (e). f Telomere count in 2D using the Find Maxima setting in ImageJ and various noise tolerances. g Telomere count in 3D using Imaris ‘Spots’ feature. Hs68 + hTERT cells have on average less telomeres per cell compared to Hs68. Two-tailed unpaired t-tests were performed, n = 3 biologically independent experiments, ****P < 0.0001. Source data used for the graphs can be found in Supplementary Data 3.

    Article Snippet: Primary human foreskin fibroblasts Hs68 (CRL-1635, ATCC) cells were grown in DMEM, 1 g/L glucose (Gibco, 11885–084) containing 10% fetal bovine serum (FBS).

    Techniques: Two Tailed Test