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Deletion of the 20q-TERRA locus decreases telomere length and protection in U2OS cells. ( a ) Q-FISH images obtained from metaphases spreads from U2OS cells WT and KO for the Chr20q-TERRA locus (clones A4, B4 and C4). (Left graphs) Frequency graphs of telomere length (a.u.) distribution measured in WT and in the 20q-KO cells (clones A4, B4 and C4) from <t>three</t> independent experiments. The mean telomere length and the number of telomeres and metaphases analyzed is shown. The red lines are arbitrary lines placed in the exact same position in each frequency graph to visualize differences between the 20q-KO clones and the WT controls (right graphs) The mean telomere length, the percentage of short telomeres and the quantification of signal-free ends per metaphase are also represented. Short telomeres are considered those in the 10% percentile of the total telomere length distribution. Total number of metaphases used for the statistical analysis is indicated. Scale bar, 10 μm and (zoom) 1 μm. ( b ) WT and 20q-KO cells were analyzed for T-SCE events with G-rich (green) and C-rich (red) PNA probes. The fraction of chromosome ends with T-SCE obtained from three different experiments was quantified and graphed as the mean values±s.e.m., n =30 metaphases. The number of metaphases analyzed is shown. Only events in which interchange of both colours were quantified (see examples of no-T-SCE and T-SCE). The quantification was carried out by counting the number of events in the same chromosome or in different chromosomes and then normalizing it by the total number of chromosomes observed in each metaphase. Scale bar, 1 μm. ( c ) Quantification of <t>DNA-containing</t> double minute chromosomes (TDMs) in WT and 20q-KO cells from three different experiments (mean values±s.e.m., n =30 metaphases). An example of TDMs is shown. One-way Anova with Dunnett's post test was used for all statistical analysis (* P
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1) Product Images from "Telomeric RNAs are essential to maintain telomeres"

Article Title: Telomeric RNAs are essential to maintain telomeres

Journal: Nature Communications

doi: 10.1038/ncomms12534

Deletion of the 20q-TERRA locus decreases telomere length and protection in U2OS cells. ( a ) Q-FISH images obtained from metaphases spreads from U2OS cells WT and KO for the Chr20q-TERRA locus (clones A4, B4 and C4). (Left graphs) Frequency graphs of telomere length (a.u.) distribution measured in WT and in the 20q-KO cells (clones A4, B4 and C4) from three independent experiments. The mean telomere length and the number of telomeres and metaphases analyzed is shown. The red lines are arbitrary lines placed in the exact same position in each frequency graph to visualize differences between the 20q-KO clones and the WT controls (right graphs) The mean telomere length, the percentage of short telomeres and the quantification of signal-free ends per metaphase are also represented. Short telomeres are considered those in the 10% percentile of the total telomere length distribution. Total number of metaphases used for the statistical analysis is indicated. Scale bar, 10 μm and (zoom) 1 μm. ( b ) WT and 20q-KO cells were analyzed for T-SCE events with G-rich (green) and C-rich (red) PNA probes. The fraction of chromosome ends with T-SCE obtained from three different experiments was quantified and graphed as the mean values±s.e.m., n =30 metaphases. The number of metaphases analyzed is shown. Only events in which interchange of both colours were quantified (see examples of no-T-SCE and T-SCE). The quantification was carried out by counting the number of events in the same chromosome or in different chromosomes and then normalizing it by the total number of chromosomes observed in each metaphase. Scale bar, 1 μm. ( c ) Quantification of DNA-containing double minute chromosomes (TDMs) in WT and 20q-KO cells from three different experiments (mean values±s.e.m., n =30 metaphases). An example of TDMs is shown. One-way Anova with Dunnett's post test was used for all statistical analysis (* P
Figure Legend Snippet: Deletion of the 20q-TERRA locus decreases telomere length and protection in U2OS cells. ( a ) Q-FISH images obtained from metaphases spreads from U2OS cells WT and KO for the Chr20q-TERRA locus (clones A4, B4 and C4). (Left graphs) Frequency graphs of telomere length (a.u.) distribution measured in WT and in the 20q-KO cells (clones A4, B4 and C4) from three independent experiments. The mean telomere length and the number of telomeres and metaphases analyzed is shown. The red lines are arbitrary lines placed in the exact same position in each frequency graph to visualize differences between the 20q-KO clones and the WT controls (right graphs) The mean telomere length, the percentage of short telomeres and the quantification of signal-free ends per metaphase are also represented. Short telomeres are considered those in the 10% percentile of the total telomere length distribution. Total number of metaphases used for the statistical analysis is indicated. Scale bar, 10 μm and (zoom) 1 μm. ( b ) WT and 20q-KO cells were analyzed for T-SCE events with G-rich (green) and C-rich (red) PNA probes. The fraction of chromosome ends with T-SCE obtained from three different experiments was quantified and graphed as the mean values±s.e.m., n =30 metaphases. The number of metaphases analyzed is shown. Only events in which interchange of both colours were quantified (see examples of no-T-SCE and T-SCE). The quantification was carried out by counting the number of events in the same chromosome or in different chromosomes and then normalizing it by the total number of chromosomes observed in each metaphase. Scale bar, 1 μm. ( c ) Quantification of DNA-containing double minute chromosomes (TDMs) in WT and 20q-KO cells from three different experiments (mean values±s.e.m., n =30 metaphases). An example of TDMs is shown. One-way Anova with Dunnett's post test was used for all statistical analysis (* P

Techniques Used: Fluorescence In Situ Hybridization, Clone Assay

Deletion of the 20q-TERRA locus decreases telomere protection in U2OS cells. ( a ) Quantification of the total γH2AX signal per nucleus (mean values±s.e.m., n =number of cells) is shown. The total number of cells analyzed is indicated. ( b ) Quantification of the total 53BP1 spot signal per nucleus (mean values±s.e.m., n =number of cells is shown). The total number of cells analyzed is indicated. ( c ) Graphs showing the quantification of the co-localization (TIF) between TRF2 and either γH2AX or 53BP1 in WT cells and in all 20q-KO clones (mean values±s.e.m., n =3 independent experiments for γH2AX and n =number of cells for 53BP1) per cell is shown. The total number of nuclei analyzed is indicated. ( d ) Representative images of the average number of TIFs found on double inmunostain to detect the telomere protein TRF2 (green) and either the DNA damage markers phospho-Histone γH2AX or 53BP1 (red) in the U2OS cells WT or deleted for the 20q locus. Arrowheads indicate co-localization events. Scale bar, 10 μm. ( e ) Quantification of chromosomal end-to-end fusions in WT and in the 20q-KO cells from three independent experiments (mean values±s.e.m., n =metaphases). Examples of end-to-end fusions are shown as well. Scale bar, 1 μm. ( f ) Array-CGH analysis was performed on hybridization on the same membrane of DNA differentially labelled from WT and 20q-KO cells. The chromosomal gains and losses in 20q-KO cells normalized by WT cells are represented. The chromosomal gains are shown in green and in red the chromosomal losses. One-way Anova with Dunnett's post test was used for all statistical analysis except for the quantification of chromosomal fusions in which the Student's t -test was used (* P
Figure Legend Snippet: Deletion of the 20q-TERRA locus decreases telomere protection in U2OS cells. ( a ) Quantification of the total γH2AX signal per nucleus (mean values±s.e.m., n =number of cells) is shown. The total number of cells analyzed is indicated. ( b ) Quantification of the total 53BP1 spot signal per nucleus (mean values±s.e.m., n =number of cells is shown). The total number of cells analyzed is indicated. ( c ) Graphs showing the quantification of the co-localization (TIF) between TRF2 and either γH2AX or 53BP1 in WT cells and in all 20q-KO clones (mean values±s.e.m., n =3 independent experiments for γH2AX and n =number of cells for 53BP1) per cell is shown. The total number of nuclei analyzed is indicated. ( d ) Representative images of the average number of TIFs found on double inmunostain to detect the telomere protein TRF2 (green) and either the DNA damage markers phospho-Histone γH2AX or 53BP1 (red) in the U2OS cells WT or deleted for the 20q locus. Arrowheads indicate co-localization events. Scale bar, 10 μm. ( e ) Quantification of chromosomal end-to-end fusions in WT and in the 20q-KO cells from three independent experiments (mean values±s.e.m., n =metaphases). Examples of end-to-end fusions are shown as well. Scale bar, 1 μm. ( f ) Array-CGH analysis was performed on hybridization on the same membrane of DNA differentially labelled from WT and 20q-KO cells. The chromosomal gains and losses in 20q-KO cells normalized by WT cells are represented. The chromosomal gains are shown in green and in red the chromosomal losses. One-way Anova with Dunnett's post test was used for all statistical analysis except for the quantification of chromosomal fusions in which the Student's t -test was used (* P

Techniques Used: Clone Assay, Hybridization

2) Product Images from "Asynchronous replication timing of telomeres at opposite arms of mammalian chromosomes"

Article Title: Asynchronous replication timing of telomeres at opposite arms of mammalian chromosomes

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.0404106101

Schema of ReDFISH technique. ( a ) ReDFISH of a chromosome that has replicated fully in the presence of BrdUrd/dC. Newly synthesized DNA incorporating BrdUrd/dC (horizontal stripes) is removed after nicking the DNA with Hoechst 33258 plus UV and digesting nicked DNA with exonuclease III, leaving only the parental strands. The G-rich telomeric strand is the template for lagging strand synthesis and anneals to a Cy3-conjugated C-rich telomeric probe, whereas the C-rich telomeric strand is the template for leading strand synthesis and anneals to an FITC-conjugated G-rich telomeric probe. This pattern defines which telomeric strands replicated at the time of BrdUrd/BrdC labeling. ( b ) ReDFISH of a partially replicated chromosome. In this example, only the p-arm telomere of the X chromosome was replicating during the 1-h BrdUrd/dC pulse; the q arm was not replicating. As a consequence, only the parental strands are available for hybridization in the p arm (schema, p arms), whereas both strands of unreplicated q-arm DNA survive digestion and hybridize to both probes (schema, q arms).
Figure Legend Snippet: Schema of ReDFISH technique. ( a ) ReDFISH of a chromosome that has replicated fully in the presence of BrdUrd/dC. Newly synthesized DNA incorporating BrdUrd/dC (horizontal stripes) is removed after nicking the DNA with Hoechst 33258 plus UV and digesting nicked DNA with exonuclease III, leaving only the parental strands. The G-rich telomeric strand is the template for lagging strand synthesis and anneals to a Cy3-conjugated C-rich telomeric probe, whereas the C-rich telomeric strand is the template for leading strand synthesis and anneals to an FITC-conjugated G-rich telomeric probe. This pattern defines which telomeric strands replicated at the time of BrdUrd/BrdC labeling. ( b ) ReDFISH of a partially replicated chromosome. In this example, only the p-arm telomere of the X chromosome was replicating during the 1-h BrdUrd/dC pulse; the q arm was not replicating. As a consequence, only the parental strands are available for hybridization in the p arm (schema, p arms), whereas both strands of unreplicated q-arm DNA survive digestion and hybridize to both probes (schema, q arms).

Techniques Used: Synthesized, Labeling, Hybridization

3) Product Images from "cis and trans Requirements for Rolling Circle Replication of a Satellite RNA"

Article Title: cis and trans Requirements for Rolling Circle Replication of a Satellite RNA

Journal: Journal of Virology

doi: 10.1128/JVI.78.6.3072-3082.2004

Secondary structure of satRPV RNA monomer. (A) Autoradiograph of 5′-end-labeled transcript of monomeric satRPV RNA after partial digestion with imidazole or RNase T 1 . Gel-purified, end-labeled RNA was incubated in three different molarities of imidazole under nondenaturing (native) conditions for 15 h (lanes 3 to 6). Indicated units of RNase T 1 were used for digestion in nondenaturing (native) conditions for 5 min at 25°C. To generate the G-track sequencing ladder, indicated units of RNase T 1 ) superimposed with markers indicating intensity of cleavages in panel A. Open, filled, and double symbols indicate weak, moderate, and strong cuts, respectively.
Figure Legend Snippet: Secondary structure of satRPV RNA monomer. (A) Autoradiograph of 5′-end-labeled transcript of monomeric satRPV RNA after partial digestion with imidazole or RNase T 1 . Gel-purified, end-labeled RNA was incubated in three different molarities of imidazole under nondenaturing (native) conditions for 15 h (lanes 3 to 6). Indicated units of RNase T 1 were used for digestion in nondenaturing (native) conditions for 5 min at 25°C. To generate the G-track sequencing ladder, indicated units of RNase T 1 ) superimposed with markers indicating intensity of cleavages in panel A. Open, filled, and double symbols indicate weak, moderate, and strong cuts, respectively.

Techniques Used: Autoradiography, Labeling, Purification, Incubation, Sequencing

4) Product Images from "Noninvasive gene targeting to the brain"

Article Title: Noninvasive gene targeting to the brain

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi:

β-galactosidase histochemistry in brain ( A – E ) and liver ( F ) at 48 h after i.v. injection of the β-galactosidase gene packaged inside the OX26 pegylated immunoliposome ( B – F ). The control brain from rats receiving no gene administration is shown in B . Magnification bars = 1.5 mm ( A ), 2.2 mm ( B ), 57 μm ( C ), 23 μm ( D ), 230 μm ( E ), and 15 μm ( F ). A and B were not counterstained. The lateral ventricles (LV), third ventricle (III), left or right hippocampus (hippo), and hypothalamic supraoptic nuclei (son) are labeled in A . C shows punctate gene expression in intraparenchymal capillaries and may represent gene expression in either endothelium or microvascular pericytes. ( D ) Gene expression in the epithelium of the choroid plexus. The lumen (L) of a capillary of the choroid plexus is shown and demonstrates the absence of β-galactosidase enzyme activity in the plasma compartment. ( E ) The thalamic (thal) nuclei below the choroid plexus of the third ventricle, which is also visible in A . ( F ) Abundant gene expression in hepatocytes; this high magnification shows a speckled pattern, suggesting localization of the β-galactosidase enzyme within the liver cell endoplasmic reticulum.
Figure Legend Snippet: β-galactosidase histochemistry in brain ( A – E ) and liver ( F ) at 48 h after i.v. injection of the β-galactosidase gene packaged inside the OX26 pegylated immunoliposome ( B – F ). The control brain from rats receiving no gene administration is shown in B . Magnification bars = 1.5 mm ( A ), 2.2 mm ( B ), 57 μm ( C ), 23 μm ( D ), 230 μm ( E ), and 15 μm ( F ). A and B were not counterstained. The lateral ventricles (LV), third ventricle (III), left or right hippocampus (hippo), and hypothalamic supraoptic nuclei (son) are labeled in A . C shows punctate gene expression in intraparenchymal capillaries and may represent gene expression in either endothelium or microvascular pericytes. ( D ) Gene expression in the epithelium of the choroid plexus. The lumen (L) of a capillary of the choroid plexus is shown and demonstrates the absence of β-galactosidase enzyme activity in the plasma compartment. ( E ) The thalamic (thal) nuclei below the choroid plexus of the third ventricle, which is also visible in A . ( F ) Abundant gene expression in hepatocytes; this high magnification shows a speckled pattern, suggesting localization of the β-galactosidase enzyme within the liver cell endoplasmic reticulum.

Techniques Used: Injection, Labeling, Expressing, Activity Assay

5) Product Images from "Functional interaction between DNA-PKcs and telomerase in telomere length maintenance"

Article Title: Functional interaction between DNA-PKcs and telomerase in telomere length maintenance

Journal: The EMBO Journal

doi: 10.1093/emboj/cdf593

Fig. 2. Telomere length determination using Q-FISH on testis sections. ( A ) Average telomere fluorescence in arbitrary units (a.u.f.) of 100 meiotic cells from each mouse of the indicated genotype. Standard deviation, as well as the total number of meiocytes analyzed per mouse are indicated. ( B ) Average telomere fluorescence in arbitrary units (a.u.f.) of three or four mice grouped per genotype. Standard deviation, as well as the total number of mice of each genotype, are indicated. There was a significant decrease in average telomere fluorescence in G1 Terc –/– /DNA-PKcs –/– meiotic cells compared with G1 Terc –/– /DNA-PKcs +/+ controls ( P
Figure Legend Snippet: Fig. 2. Telomere length determination using Q-FISH on testis sections. ( A ) Average telomere fluorescence in arbitrary units (a.u.f.) of 100 meiotic cells from each mouse of the indicated genotype. Standard deviation, as well as the total number of meiocytes analyzed per mouse are indicated. ( B ) Average telomere fluorescence in arbitrary units (a.u.f.) of three or four mice grouped per genotype. Standard deviation, as well as the total number of mice of each genotype, are indicated. There was a significant decrease in average telomere fluorescence in G1 Terc –/– /DNA-PKcs –/– meiotic cells compared with G1 Terc –/– /DNA-PKcs +/+ controls ( P

Techniques Used: Fluorescence In Situ Hybridization, Fluorescence, Standard Deviation, Mouse Assay

6) Product Images from "Alternative lengthening of human telomeres is a conservative DNA replication process with features of break‐induced replication"

Article Title: Alternative lengthening of human telomeres is a conservative DNA replication process with features of break‐induced replication

Journal: EMBO Reports

doi: 10.15252/embr.201643169

Conservative DNA replication at telomeres of human ALT cells Examples of chromosome arms exhibiting features of telomeric semiconservative (Semi) replication, conservative (Consrv) replication of the entire telomere (E) and conservative replication of part of the telomere (P). Idealized chromosome diagrams (left) and actual microscopy images (right) are shown. D‐FISH, denaturing FISH; ND‐CO‐FISH, non‐denaturing CO‐FISH. Percentages of chromosome arms exhibiting conservative (Consrv) replication of the entire telomere (E), conservative replication of part of the telomere (P), and conservative replication of either the entire telomere of part of it (E+P). NE, U2OS cells expressing normal levels of cyclin E; OE, U2OS cells overexpressing cyclin E for 4 days. Bars represent means and standard errors of the mean from three independent experiments. Cyclin E overexpression resulted in higher percentages of conservatively replicated telomeres: P
Figure Legend Snippet: Conservative DNA replication at telomeres of human ALT cells Examples of chromosome arms exhibiting features of telomeric semiconservative (Semi) replication, conservative (Consrv) replication of the entire telomere (E) and conservative replication of part of the telomere (P). Idealized chromosome diagrams (left) and actual microscopy images (right) are shown. D‐FISH, denaturing FISH; ND‐CO‐FISH, non‐denaturing CO‐FISH. Percentages of chromosome arms exhibiting conservative (Consrv) replication of the entire telomere (E), conservative replication of part of the telomere (P), and conservative replication of either the entire telomere of part of it (E+P). NE, U2OS cells expressing normal levels of cyclin E; OE, U2OS cells overexpressing cyclin E for 4 days. Bars represent means and standard errors of the mean from three independent experiments. Cyclin E overexpression resulted in higher percentages of conservatively replicated telomeres: P

Techniques Used: Microscopy, Fluorescence In Situ Hybridization, Expressing, Over Expression

Triple‐FISH protocol to distinguish between telomeric semiconservative and conservative DNA replication Semiconservative replication. Diagram of a chromosome with the telomeric C‐rich and G‐rich strands colored red and green, respectively. Newly synthesized strands are indicated by dotted lines. The three steps of the protocol were strand‐specific: dual color, denaturing FISH (1), non‐denaturing chromosome orientation (CO)‐FISH (2), and denaturing FISH (3). In the second step, the nascent strands have been digested. In all steps, two sets of PNA primers specific for the G‐strand and C‐strand, respectively, were used to monitor the presence of both strands. The arrows indicate the color of the emitted light and its intensity (idealized). Conservative replication. Diagram showing that telomeric conservative replication (shown here to involve the entire length of the telomeres at the p arms) leads to distinct staining patterns, not observed with semiconservative replication. BIR, break‐induced replication. Partially conservative and partially semiconservative telomeric replication. Diagram showing the staining patterns predicted for telomeres that are partially conservatively replicated (distal half) and partially semiconservatively replicated (proximal half), as might occur following fork collapse within a telomere (shown only for the telomeres at the p arms).
Figure Legend Snippet: Triple‐FISH protocol to distinguish between telomeric semiconservative and conservative DNA replication Semiconservative replication. Diagram of a chromosome with the telomeric C‐rich and G‐rich strands colored red and green, respectively. Newly synthesized strands are indicated by dotted lines. The three steps of the protocol were strand‐specific: dual color, denaturing FISH (1), non‐denaturing chromosome orientation (CO)‐FISH (2), and denaturing FISH (3). In the second step, the nascent strands have been digested. In all steps, two sets of PNA primers specific for the G‐strand and C‐strand, respectively, were used to monitor the presence of both strands. The arrows indicate the color of the emitted light and its intensity (idealized). Conservative replication. Diagram showing that telomeric conservative replication (shown here to involve the entire length of the telomeres at the p arms) leads to distinct staining patterns, not observed with semiconservative replication. BIR, break‐induced replication. Partially conservative and partially semiconservative telomeric replication. Diagram showing the staining patterns predicted for telomeres that are partially conservatively replicated (distal half) and partially semiconservatively replicated (proximal half), as might occur following fork collapse within a telomere (shown only for the telomeres at the p arms).

Techniques Used: Fluorescence In Situ Hybridization, Synthesized, Staining

7) Product Images from "DNA-PK-dependent binding of DNA ends to plasmids containing nuclear matrix attachment region DNA sequences: evidence for assembly of a repair complex"

Article Title: DNA-PK-dependent binding of DNA ends to plasmids containing nuclear matrix attachment region DNA sequences: evidence for assembly of a repair complex

Journal: Nucleic Acids Research

doi:

Identification of proteins preferentially associated with the pMII-MAR plasmid. Mobility shift reactions with (+Ends) or without (–Ends) unlabeled 144 bp probe were separated in a preparative 1.5% agarose gel, pUC18 and MII plasmid bands excised, and proteins electroeluted as described in Materials and Methods. Proteins eluted from equal amounts (weight) of agarose were subjected to SDS–PAGE and western blot analysis. Western blots were probed with antibodies directed against DNA-PKcs and Ku86 ( A ), XRCC4 ( B ), SAF-A ( C ), DNA ligase IV ( D ), Topoisomerase II ( E ) or PARP ( F ). Lane 5 of (A)–(F) contained 25 µg of M059K nuclear extract. For clarity, lanes in western blots (B) and (F) were rearranged; however, each blot contained proteins from the same experiment treated under identical conditions. Proteins extracted from three independent mobility shift reactions produced similar band patterns.
Figure Legend Snippet: Identification of proteins preferentially associated with the pMII-MAR plasmid. Mobility shift reactions with (+Ends) or without (–Ends) unlabeled 144 bp probe were separated in a preparative 1.5% agarose gel, pUC18 and MII plasmid bands excised, and proteins electroeluted as described in Materials and Methods. Proteins eluted from equal amounts (weight) of agarose were subjected to SDS–PAGE and western blot analysis. Western blots were probed with antibodies directed against DNA-PKcs and Ku86 ( A ), XRCC4 ( B ), SAF-A ( C ), DNA ligase IV ( D ), Topoisomerase II ( E ) or PARP ( F ). Lane 5 of (A)–(F) contained 25 µg of M059K nuclear extract. For clarity, lanes in western blots (B) and (F) were rearranged; however, each blot contained proteins from the same experiment treated under identical conditions. Proteins extracted from three independent mobility shift reactions produced similar band patterns.

Techniques Used: Plasmid Preparation, Mobility Shift, Agarose Gel Electrophoresis, SDS Page, Western Blot, Produced

8) Product Images from "Telomere Reprogramming and Maintenance in Porcine iPS Cells"

Article Title: Telomere Reprogramming and Maintenance in Porcine iPS Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0074202

DNA damage and telomere dysfunction-induced foci (TIF) in porcine iPS cells. (A, C, E and G) Percentage of γH2Ax positive cells. Cells are categorized into three groups with fewer than 10, 10–30 and more than 30 γH2Ax foci, respectively. n = number of cells counted. *p
Figure Legend Snippet: DNA damage and telomere dysfunction-induced foci (TIF) in porcine iPS cells. (A, C, E and G) Percentage of γH2Ax positive cells. Cells are categorized into three groups with fewer than 10, 10–30 and more than 30 γH2Ax foci, respectively. n = number of cells counted. *p

Techniques Used:

9) Product Images from "PRL-3 promotes telomere deprotection and chromosomal instability"

Article Title: PRL-3 promotes telomere deprotection and chromosomal instability

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkx392

Overexpression of PRL-3 promotes telomere dysfunction. ( A ) Validation of PRL-3 stable overexpression. WI38 fibroblasts were infected with control or PRL-3-expressing letivirus. Expression vectors pcDNA3-myc-PRL-3 (for HCT116 cells), pcDNA3.1-myc-PRL-3 (for LoVo cells) and the respective control plasmids were transfected into cells, followed by selection and pooling of stable colonies. Cell lysates were examined by western blot with antibodies to PRL-3, TRF2 and RAP1. ( B ) Effects of PRL-3 stable overexpression on γH2AX, pCHK1 and p53 levels. Indicated cells were treated with ETP (20 μM) or DMSO (1:1000) for 4 h. ( C ) Effects of PRL-3 stable overexpression on TIF formation. WI38 cells were analyzed by IF-FISH staining of pATM (green) and telomere (red). Left, representative staining. Arrows, foci of TIFs. Scale bar, 5 μm. Right, quantification of cells with ≥5 TIFs. Mean ± SD of two independent experiments. n > 60 metaphase per single experiment. Student's t -test. ( D ) Effects of PRL-3 stable overexpression on dysfunctional telomere repair pathways. Upper, representative CO-FISH staining of WI38 cells. Metaphase cells were stained with probes specific for leading (red) and lagging (green) strands and counterstained with DAPI (blue). Yellow arrow, a typical T-SCE. White arrow, a chromosome–chromosome fusion. Red arrowhead, a MTS. Scale bar, 2.5 μm. Lower, quantification of abnormalities. Mean ± SD of two independent experiments. n > 1300 chromosomes per single experiment. Student's t -test. ( E ) Southern blot analysis of PRL-3 stable overexpression-induced telomere deprotection. Genomic DNA from indicated cells were resolved on agarose gel, transferred to nitrocellulose membrane and probed with biotin-labeled telomere probe. ( F ) qPCR analysis of PRL-3 stable overexpression-induced telomere deprotection. Relative telomere to single copy gene (T/S) ratio of control cells was set as 1. Mean ± SD of three independent experiments. n = 4 replicates per single experiment. Student's t -test.
Figure Legend Snippet: Overexpression of PRL-3 promotes telomere dysfunction. ( A ) Validation of PRL-3 stable overexpression. WI38 fibroblasts were infected with control or PRL-3-expressing letivirus. Expression vectors pcDNA3-myc-PRL-3 (for HCT116 cells), pcDNA3.1-myc-PRL-3 (for LoVo cells) and the respective control plasmids were transfected into cells, followed by selection and pooling of stable colonies. Cell lysates were examined by western blot with antibodies to PRL-3, TRF2 and RAP1. ( B ) Effects of PRL-3 stable overexpression on γH2AX, pCHK1 and p53 levels. Indicated cells were treated with ETP (20 μM) or DMSO (1:1000) for 4 h. ( C ) Effects of PRL-3 stable overexpression on TIF formation. WI38 cells were analyzed by IF-FISH staining of pATM (green) and telomere (red). Left, representative staining. Arrows, foci of TIFs. Scale bar, 5 μm. Right, quantification of cells with ≥5 TIFs. Mean ± SD of two independent experiments. n > 60 metaphase per single experiment. Student's t -test. ( D ) Effects of PRL-3 stable overexpression on dysfunctional telomere repair pathways. Upper, representative CO-FISH staining of WI38 cells. Metaphase cells were stained with probes specific for leading (red) and lagging (green) strands and counterstained with DAPI (blue). Yellow arrow, a typical T-SCE. White arrow, a chromosome–chromosome fusion. Red arrowhead, a MTS. Scale bar, 2.5 μm. Lower, quantification of abnormalities. Mean ± SD of two independent experiments. n > 1300 chromosomes per single experiment. Student's t -test. ( E ) Southern blot analysis of PRL-3 stable overexpression-induced telomere deprotection. Genomic DNA from indicated cells were resolved on agarose gel, transferred to nitrocellulose membrane and probed with biotin-labeled telomere probe. ( F ) qPCR analysis of PRL-3 stable overexpression-induced telomere deprotection. Relative telomere to single copy gene (T/S) ratio of control cells was set as 1. Mean ± SD of three independent experiments. n = 4 replicates per single experiment. Student's t -test.

Techniques Used: Over Expression, Infection, Expressing, Transfection, Selection, Western Blot, Fluorescence In Situ Hybridization, Staining, Southern Blot, Agarose Gel Electrophoresis, Labeling, Real-time Polymerase Chain Reaction

Overexpression of PRL-3 promotes chromosomal instability and senescence. ( A ) Effects of PRL-3 stable overexpression on APB and MN formation. Indicated cells were treated with aphidicolin (0.2 μM) or DMSO (1:1000) for 24 h, followed by DAPI staining. Mean ± SD of two independent experiments. Student's t -test. n > 1500 cells scored per sample for MN or n > 60 anaphase cells scored per sample for APB. ( B ) Effects of PRL-3 stable overexpression on BrdU incorporation. Indicated cells were treated with double-thymidine block, released into fresh medium containing 10 μM BrdU and incubated for 45 min. Cells were fixed, immunostained with anti-BrdU (green), and counterstained with DAPI (blue). Left, representative staining of BrdU. Scale bar, 15 μm. Right, quantification of BrdU-positive cells. Mean ± SD of two independent experiments. n > 300 cells per single experiment. Student's t -test. ( C ) Effects of PRL-3 stable overexpression on senescence. Indicated cells were treated with DMSO (1:1000) or Ku55933 (5 μM) for 24 h, followed by β-galactosidase staining. Left, representative staining. Right, quantification of β-galactosidase positive cells. Mean ± SD of three independent experiments. n > 500 cells per single experiment. Student's t -test. ( D ) Effects of PRL-3 stable overexpression on H3K9me3 levels. Indicated cells were fixed, immunostained with anti-H3K9me3 (red), and counterstained with DAPI (blue). ( E ) Effects of reconstituted PRL-3 on telomere length, DNA damage and senescence in PRL-3 stable knockdown cells. HCT116 control and PRL-3 stable knockdown cells were co-transfected with indicated amount of pcDNA3 and pcDNA3-PRL-3 plasmids. The total amount of plasmids for each sample was adjusted to 4 μg. After 72 h, protein lysates were subjected to western blot of PRL-3, γH2AX, H3K9me3 (lower). Genomic DNA was used for qPCR analysis of telomere length (upper). Protein bands were scanned and relative OD was calculated by normalizing to GAPDH. T/S ratio of HCT116 control cells transfected with pcDNA3 was set as 1. Pearson χ2 test.
Figure Legend Snippet: Overexpression of PRL-3 promotes chromosomal instability and senescence. ( A ) Effects of PRL-3 stable overexpression on APB and MN formation. Indicated cells were treated with aphidicolin (0.2 μM) or DMSO (1:1000) for 24 h, followed by DAPI staining. Mean ± SD of two independent experiments. Student's t -test. n > 1500 cells scored per sample for MN or n > 60 anaphase cells scored per sample for APB. ( B ) Effects of PRL-3 stable overexpression on BrdU incorporation. Indicated cells were treated with double-thymidine block, released into fresh medium containing 10 μM BrdU and incubated for 45 min. Cells were fixed, immunostained with anti-BrdU (green), and counterstained with DAPI (blue). Left, representative staining of BrdU. Scale bar, 15 μm. Right, quantification of BrdU-positive cells. Mean ± SD of two independent experiments. n > 300 cells per single experiment. Student's t -test. ( C ) Effects of PRL-3 stable overexpression on senescence. Indicated cells were treated with DMSO (1:1000) or Ku55933 (5 μM) for 24 h, followed by β-galactosidase staining. Left, representative staining. Right, quantification of β-galactosidase positive cells. Mean ± SD of three independent experiments. n > 500 cells per single experiment. Student's t -test. ( D ) Effects of PRL-3 stable overexpression on H3K9me3 levels. Indicated cells were fixed, immunostained with anti-H3K9me3 (red), and counterstained with DAPI (blue). ( E ) Effects of reconstituted PRL-3 on telomere length, DNA damage and senescence in PRL-3 stable knockdown cells. HCT116 control and PRL-3 stable knockdown cells were co-transfected with indicated amount of pcDNA3 and pcDNA3-PRL-3 plasmids. The total amount of plasmids for each sample was adjusted to 4 μg. After 72 h, protein lysates were subjected to western blot of PRL-3, γH2AX, H3K9me3 (lower). Genomic DNA was used for qPCR analysis of telomere length (upper). Protein bands were scanned and relative OD was calculated by normalizing to GAPDH. T/S ratio of HCT116 control cells transfected with pcDNA3 was set as 1. Pearson χ2 test.

Techniques Used: Over Expression, Staining, BrdU Incorporation Assay, Blocking Assay, Incubation, Transfection, Western Blot, Real-time Polymerase Chain Reaction

Silencing of PRL-3 promotes DDR and senescence. ( A ) Efficiencies of PRL-3 silencing in HCT116 (knockdown by two shRNAs using lentivirus system, left) and SW480 (knockout by CRISPR/Cas9 system, right) cells and its effects on indicated protein levels. WT, wild-type. KO, knockout. ( B ) Effects of PRL-3 silencing on phosphorylations of H2AX and CHK1. Samples treated with 20 μM etoposide (ETP) for 4 h were used as positive controls. ( C ) Effects of PRL-3 silencing on TIF formation. Indicated HCT116 cells were subjected to IF-FISH staining. Upper, representative staining. Arrows, colocalizations between γH2AX and telomere (TIFs). Scale bar, 5 μm. Lower, quantification of cells with ≥5 TIF. Mean ± SD of two independent experiments. n > 200 cells per single experiment. Student's t -test. ( D ) Effects of PRL-3 silencing on anaphase bridges (APB) and micronuclei (MN) formation. Indicated cells were treated with aphidicolin (0.2 μM) or DMSO (1:1000) for 24 h, followed by DAPI staining. Mean ± SD of two independent experiments. n > 1000 cells scored per sample for MN and n > 50 anaphase cells scored per sample for APB. Student's t -test. Representative images of APB (red arrow) and MN (white arrow) of HCT116 cells stained with DAPI were shown. ( E ) ChIP analysis of RAP1 and TRF2's binding to telomeric or Alu DNA in HCT116 and S480 cells silenced for PRL-3. Upper, representative blots after ChIP with indicated antibodies or IgG. Input, 2% DNA. Lower, quantification of relative OD. Relative OD was calculated by normalizing to that of input and relative OD of control was set as 100%. Mean ± SD of three independent experiments. Student's t -test. ( F ) PRL-3 silencing induced ROS-dependent cellular senescence and DNA damage response. Indica ted HCT116 cells were treated with NAC (10 mM), GSH (10 mM) or DMSO (1:1000) for 24 h. Part of cells were fixed and processed for β-galactosidase staining, others were analyzed by western blot. Upper, representative β-galactosidase staining of cells treated with DMSO. Middle, quantification of β-galactosidase positive cells. Mean ± SD of two independent experiments. n > 400 cells per single experiment. Student's t -test. Lower, western blot of γH2AX.
Figure Legend Snippet: Silencing of PRL-3 promotes DDR and senescence. ( A ) Efficiencies of PRL-3 silencing in HCT116 (knockdown by two shRNAs using lentivirus system, left) and SW480 (knockout by CRISPR/Cas9 system, right) cells and its effects on indicated protein levels. WT, wild-type. KO, knockout. ( B ) Effects of PRL-3 silencing on phosphorylations of H2AX and CHK1. Samples treated with 20 μM etoposide (ETP) for 4 h were used as positive controls. ( C ) Effects of PRL-3 silencing on TIF formation. Indicated HCT116 cells were subjected to IF-FISH staining. Upper, representative staining. Arrows, colocalizations between γH2AX and telomere (TIFs). Scale bar, 5 μm. Lower, quantification of cells with ≥5 TIF. Mean ± SD of two independent experiments. n > 200 cells per single experiment. Student's t -test. ( D ) Effects of PRL-3 silencing on anaphase bridges (APB) and micronuclei (MN) formation. Indicated cells were treated with aphidicolin (0.2 μM) or DMSO (1:1000) for 24 h, followed by DAPI staining. Mean ± SD of two independent experiments. n > 1000 cells scored per sample for MN and n > 50 anaphase cells scored per sample for APB. Student's t -test. Representative images of APB (red arrow) and MN (white arrow) of HCT116 cells stained with DAPI were shown. ( E ) ChIP analysis of RAP1 and TRF2's binding to telomeric or Alu DNA in HCT116 and S480 cells silenced for PRL-3. Upper, representative blots after ChIP with indicated antibodies or IgG. Input, 2% DNA. Lower, quantification of relative OD. Relative OD was calculated by normalizing to that of input and relative OD of control was set as 100%. Mean ± SD of three independent experiments. Student's t -test. ( F ) PRL-3 silencing induced ROS-dependent cellular senescence and DNA damage response. Indica ted HCT116 cells were treated with NAC (10 mM), GSH (10 mM) or DMSO (1:1000) for 24 h. Part of cells were fixed and processed for β-galactosidase staining, others were analyzed by western blot. Upper, representative β-galactosidase staining of cells treated with DMSO. Middle, quantification of β-galactosidase positive cells. Mean ± SD of two independent experiments. n > 400 cells per single experiment. Student's t -test. Lower, western blot of γH2AX.

Techniques Used: Knock-Out, CRISPR, Fluorescence In Situ Hybridization, Staining, Chromatin Immunoprecipitation, Binding Assay, Western Blot

RAP1 and TRF2-dependent recruitment of PRL-3 to telomere. ( A ) In situ PLA analysis of PRL-3's associations with RAP1 and TRF2. HCT116 cells were pre-extracted, fixed, inmunostained with indicated pairs of antibodies and probed with Duolink in situ PLA reagent. Binding foci were in red and dashed lines indicated outline of nucleus (determined by DAPI counter staining). Scale bar, 10 μm. ( B ) TRF2- and RAP1-dependent recruitment of PRL-3 to telomeric DNA in vitro . Purified myc-TRF2 (150 ng), His-RAP1 (120 ng), and His-PRL-3 (30 ng) were co-incubated with 1 μg biotin-labeled telomere (lanes 1–4) or Alu (lanes 5–8) probe as indicated and subjected to pull-down analysis with Streptavidin agarose. Precipitates were analyzed by western blot with antibodies to TRF2, RAP1 and PRL-3. ( C and D ) TRF2 and RAP1-dependent recruitment of PRL-3 to telomere in cells. HCT116 cells were transfected with 50 nM indicated siRNAs for 48 h, pre-extracted, fixed and subjected to IF-FISH staining. (C) Representative PRL-3 association with telomere. Scale bar, 10 μm. Areas in white squares were enlarged. (D) Quantification of cells with ≥5 associations between PRL-3 foci and telomere. Mean ± SD of three independent experiments. n > 100 cells per single experiment. Student's t -test. ( E ) Knockdown efficiencies of RAP1 and TRF2. HCT116 cells were transfected with 50 nM siRNAs against RAP1 or TRF2 for 48 h. Lysates were analyzed by western blot with indicated antibodies. ( F ) ChIP analysis of PRL-3 binding to telomeric and Alu DNA. HCT116 cells were transfected with 50 nM indicated siRNAs for 48 h and processed for ChIP using anti-PRL-3 or pre-immune IgG. Upper, representative blots of hybridization with probe to telomere or Alu. Input, 2% DNA. Lower, quantification of relative optical densities (OD). Relative OD was calculated by normalizing to OD of Input and relative OD of control siRNA-transfected sample was set as 100%. Mean ± SD of three independent experiments. Student's t -test.
Figure Legend Snippet: RAP1 and TRF2-dependent recruitment of PRL-3 to telomere. ( A ) In situ PLA analysis of PRL-3's associations with RAP1 and TRF2. HCT116 cells were pre-extracted, fixed, inmunostained with indicated pairs of antibodies and probed with Duolink in situ PLA reagent. Binding foci were in red and dashed lines indicated outline of nucleus (determined by DAPI counter staining). Scale bar, 10 μm. ( B ) TRF2- and RAP1-dependent recruitment of PRL-3 to telomeric DNA in vitro . Purified myc-TRF2 (150 ng), His-RAP1 (120 ng), and His-PRL-3 (30 ng) were co-incubated with 1 μg biotin-labeled telomere (lanes 1–4) or Alu (lanes 5–8) probe as indicated and subjected to pull-down analysis with Streptavidin agarose. Precipitates were analyzed by western blot with antibodies to TRF2, RAP1 and PRL-3. ( C and D ) TRF2 and RAP1-dependent recruitment of PRL-3 to telomere in cells. HCT116 cells were transfected with 50 nM indicated siRNAs for 48 h, pre-extracted, fixed and subjected to IF-FISH staining. (C) Representative PRL-3 association with telomere. Scale bar, 10 μm. Areas in white squares were enlarged. (D) Quantification of cells with ≥5 associations between PRL-3 foci and telomere. Mean ± SD of three independent experiments. n > 100 cells per single experiment. Student's t -test. ( E ) Knockdown efficiencies of RAP1 and TRF2. HCT116 cells were transfected with 50 nM siRNAs against RAP1 or TRF2 for 48 h. Lysates were analyzed by western blot with indicated antibodies. ( F ) ChIP analysis of PRL-3 binding to telomeric and Alu DNA. HCT116 cells were transfected with 50 nM indicated siRNAs for 48 h and processed for ChIP using anti-PRL-3 or pre-immune IgG. Upper, representative blots of hybridization with probe to telomere or Alu. Input, 2% DNA. Lower, quantification of relative optical densities (OD). Relative OD was calculated by normalizing to OD of Input and relative OD of control siRNA-transfected sample was set as 100%. Mean ± SD of three independent experiments. Student's t -test.

Techniques Used: In Situ, Proximity Ligation Assay, Binding Assay, Staining, In Vitro, Purification, Incubation, Labeling, Western Blot, Transfection, Fluorescence In Situ Hybridization, Chromatin Immunoprecipitation, Hybridization

PRL-3 relocates RAP1 and TRF2 from telomeric DNA. ( A ) Effects of PRL-3 stable overexpression on the chromatin abundance of RAP1, TRF2 and TRF1. Nuclei from HCT116 cells were homogenized in buffer containing indicated concentrations of NaCl. Chromatin-enriched fractions were analyzed by western blot. Left, representative blots. Right, relative levels of TRF2, RAP1 and TRF1. Protein band were scanned and relative OD was calculated by normalizing to OD of H2B. The relative OD of sample prepared with 150 mM NaCl was set as 100%. Mean ± SD of three independent experiments. ANOVA. ( B ) Effects of PRL-3 stable overexpression on bindings of RAP1 and TRF2 to telomeric and Alu DNA. Indicated cells were crosslinked, immunoprecipitated with antibodies to RAP1, TRF2 or pre-immune IgG, and precipitated DNA was analyzed by ChIP. Upper, representative blots. Lower, quantification of relative OD, which was calculated by normalizing to that of Input. Relative OD of control was set as 100%. Mean ± SD of three independent experiments. Student's t -test. ( C ) Effects of PRL-3 stable overexpression on telomere associations of RAP1 and TRF2 in WI38 cells. Left, representative IF-FISH staining of telomere (red) and RAP1 or TRF2 (green). Arrows, foci of co-localization. Scale bar, 10 μm. Right, quantification of cells with ≥5 associations between RAP1 or TRF2 foci and telomere. Mean ± SD of two independent experiments. n > 80 cells per single experiment. Student's t -test. ( D ) EMSA analysis of PRL-3, RAP1 and TRF2's associations with telomeric DNA. Indicated concentrations of purified FLAG-TRF2, His-RAP1, myc-PRL-3 were co-incubated with Biotin-labeled telomere probe (20 nM). To induce super-shift, 0.1 μg anti-PRL-3 (lane 5), anti-TRF2 (lanes 6 and 18) and IgG (lane 7) were used. Note that anti-PRL-3 and anti-TRF2-induced super-shifts of Complex II partially co-migrated with Complex I (lanes 5 and 6).
Figure Legend Snippet: PRL-3 relocates RAP1 and TRF2 from telomeric DNA. ( A ) Effects of PRL-3 stable overexpression on the chromatin abundance of RAP1, TRF2 and TRF1. Nuclei from HCT116 cells were homogenized in buffer containing indicated concentrations of NaCl. Chromatin-enriched fractions were analyzed by western blot. Left, representative blots. Right, relative levels of TRF2, RAP1 and TRF1. Protein band were scanned and relative OD was calculated by normalizing to OD of H2B. The relative OD of sample prepared with 150 mM NaCl was set as 100%. Mean ± SD of three independent experiments. ANOVA. ( B ) Effects of PRL-3 stable overexpression on bindings of RAP1 and TRF2 to telomeric and Alu DNA. Indicated cells were crosslinked, immunoprecipitated with antibodies to RAP1, TRF2 or pre-immune IgG, and precipitated DNA was analyzed by ChIP. Upper, representative blots. Lower, quantification of relative OD, which was calculated by normalizing to that of Input. Relative OD of control was set as 100%. Mean ± SD of three independent experiments. Student's t -test. ( C ) Effects of PRL-3 stable overexpression on telomere associations of RAP1 and TRF2 in WI38 cells. Left, representative IF-FISH staining of telomere (red) and RAP1 or TRF2 (green). Arrows, foci of co-localization. Scale bar, 10 μm. Right, quantification of cells with ≥5 associations between RAP1 or TRF2 foci and telomere. Mean ± SD of two independent experiments. n > 80 cells per single experiment. Student's t -test. ( D ) EMSA analysis of PRL-3, RAP1 and TRF2's associations with telomeric DNA. Indicated concentrations of purified FLAG-TRF2, His-RAP1, myc-PRL-3 were co-incubated with Biotin-labeled telomere probe (20 nM). To induce super-shift, 0.1 μg anti-PRL-3 (lane 5), anti-TRF2 (lanes 6 and 18) and IgG (lane 7) were used. Note that anti-PRL-3 and anti-TRF2-induced super-shifts of Complex II partially co-migrated with Complex I (lanes 5 and 6).

Techniques Used: Over Expression, Western Blot, Immunoprecipitation, Chromatin Immunoprecipitation, Fluorescence In Situ Hybridization, Staining, Purification, Incubation, Labeling

Disrupting PRL-3-RAP1 complex or expressing ectopic TRF2 attenuates PRL-3 overexpression-promoted telomere deprotection, DNA damage, chromosomal instability and senescence. ( A ) HCT116 control and PRL-3 overexpressing cells were transfected with 0.5 μg of pEGFP-N1-Myb or pEGFP-N1 plasmid for 72 h, and indicated proteins were analyzed by western blot. ( B ) qPCR analysis of telomere length of cells in (A). T/S ratio of HCT116 control cells transfected with pEGFP-N1 was set as 1. Mean ± SD of three independent experiments. Three replicates per single experiment. Student's t -test. ( C ) Quantification of micronuclei of cells in (A). Mean ± SD of two independent experiments. n > 500 cells per single experiment. Student's t -test. ( D ) Quantification of β-galactosidase-positive cells in (A). Mean ± SD of two independent experiments. n > 300 cells per single experiment. Student's t -test. ( E ) Relative migration of cells in (A). Cells were allowed to migrate through transwell chambers for 24 h. Value of HCT116 control cells transfected with pEGFP-N1 was set as 1. Mean ± SD of two independent experiments. Three replicates per single experiment. Student's t -test. ( F ) HCT116 control and PRL-3 overexpressing cells were infected with control (Lv-con) or TRF2-expressing lentivirus (Lv-TRF2) for 120 h, and lysates were subjected to western blot. ( G ) qPCR analysis of telomere length of cells in (F). T/S ratio of HCT116 control cells infected with Lv-con was set as 1. Mean ± SD of three independent experiments. 3 replicates per single experiment. Student's t -test. ( H ) Quantification of micronuclei of cells in (F). Mean ± SD of three independent experiments. n > 500 cells per single experiment. Student's t -test. ( I ) Quantification of β-galactosidase-positive cells in (F). Mean ± SD of three independent experiments. n > 300 cells per single experiment. Student's t -test. ( J ) Relative migration of cells of (F). Cells were allowed to migrate through transwell chambers for 24 h. Value of HCT116 control cells infected with Lv-con was set as 1. Mean ± SD of three independent experiments. Three replicates per single experiment. Student's t -test.
Figure Legend Snippet: Disrupting PRL-3-RAP1 complex or expressing ectopic TRF2 attenuates PRL-3 overexpression-promoted telomere deprotection, DNA damage, chromosomal instability and senescence. ( A ) HCT116 control and PRL-3 overexpressing cells were transfected with 0.5 μg of pEGFP-N1-Myb or pEGFP-N1 plasmid for 72 h, and indicated proteins were analyzed by western blot. ( B ) qPCR analysis of telomere length of cells in (A). T/S ratio of HCT116 control cells transfected with pEGFP-N1 was set as 1. Mean ± SD of three independent experiments. Three replicates per single experiment. Student's t -test. ( C ) Quantification of micronuclei of cells in (A). Mean ± SD of two independent experiments. n > 500 cells per single experiment. Student's t -test. ( D ) Quantification of β-galactosidase-positive cells in (A). Mean ± SD of two independent experiments. n > 300 cells per single experiment. Student's t -test. ( E ) Relative migration of cells in (A). Cells were allowed to migrate through transwell chambers for 24 h. Value of HCT116 control cells transfected with pEGFP-N1 was set as 1. Mean ± SD of two independent experiments. Three replicates per single experiment. Student's t -test. ( F ) HCT116 control and PRL-3 overexpressing cells were infected with control (Lv-con) or TRF2-expressing lentivirus (Lv-TRF2) for 120 h, and lysates were subjected to western blot. ( G ) qPCR analysis of telomere length of cells in (F). T/S ratio of HCT116 control cells infected with Lv-con was set as 1. Mean ± SD of three independent experiments. 3 replicates per single experiment. Student's t -test. ( H ) Quantification of micronuclei of cells in (F). Mean ± SD of three independent experiments. n > 500 cells per single experiment. Student's t -test. ( I ) Quantification of β-galactosidase-positive cells in (F). Mean ± SD of three independent experiments. n > 300 cells per single experiment. Student's t -test. ( J ) Relative migration of cells of (F). Cells were allowed to migrate through transwell chambers for 24 h. Value of HCT116 control cells infected with Lv-con was set as 1. Mean ± SD of three independent experiments. Three replicates per single experiment. Student's t -test.

Techniques Used: Expressing, Over Expression, Transfection, Plasmid Preparation, Western Blot, Real-time Polymerase Chain Reaction, Migration, Infection

10) Product Images from "Human RECQL1 participates in telomere maintenance"

Article Title: Human RECQL1 participates in telomere maintenance

Journal: Nucleic Acids Research

doi: 10.1093/nar/gku200

RECQL1 associates to telomeres in ALT cells. ( A ) Telomeric ChIP was performed in U2OS cells using anti-RECQL1 antibody in the presence and absence of HU (treated with 5 mM for 18 h). The precipitated DNA was hybridized with the sty-11 telomeric probe (top) or an Alu probe (bottom). Dot blots were performed using anti-RECQL1 antibody, normal IgG and anti-TRF1 antibody that is used as a positive control. ( B ) Telomeric ChIP samples were confirmed by western blotting for the presence of RECQL1 and TRF1. ( C ) The graph showing the signal/input ratio from the RECQL1 IPs as a function of the quantity of chromatin (input). The results were visualized by Phosphor Imager and quantitated with Image Quant software (Molecular Dynamics) and are expressed in % binding to the telomeres. The error bars represent the mean and standard deviation from three independent ChIP assays.
Figure Legend Snippet: RECQL1 associates to telomeres in ALT cells. ( A ) Telomeric ChIP was performed in U2OS cells using anti-RECQL1 antibody in the presence and absence of HU (treated with 5 mM for 18 h). The precipitated DNA was hybridized with the sty-11 telomeric probe (top) or an Alu probe (bottom). Dot blots were performed using anti-RECQL1 antibody, normal IgG and anti-TRF1 antibody that is used as a positive control. ( B ) Telomeric ChIP samples were confirmed by western blotting for the presence of RECQL1 and TRF1. ( C ) The graph showing the signal/input ratio from the RECQL1 IPs as a function of the quantity of chromatin (input). The results were visualized by Phosphor Imager and quantitated with Image Quant software (Molecular Dynamics) and are expressed in % binding to the telomeres. The error bars represent the mean and standard deviation from three independent ChIP assays.

Techniques Used: Chromatin Immunoprecipitation, Positive Control, Western Blot, Software, Binding Assay, Standard Deviation

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    Promega exonuclease iii
    Deletion of the 20q-TERRA locus decreases telomere length and protection in U2OS cells. ( a ) Q-FISH images obtained from metaphases spreads from U2OS cells WT and KO for the Chr20q-TERRA locus (clones A4, B4 and C4). (Left graphs) Frequency graphs of telomere length (a.u.) distribution measured in WT and in the 20q-KO cells (clones A4, B4 and C4) from <t>three</t> independent experiments. The mean telomere length and the number of telomeres and metaphases analyzed is shown. The red lines are arbitrary lines placed in the exact same position in each frequency graph to visualize differences between the 20q-KO clones and the WT controls (right graphs) The mean telomere length, the percentage of short telomeres and the quantification of signal-free ends per metaphase are also represented. Short telomeres are considered those in the 10% percentile of the total telomere length distribution. Total number of metaphases used for the statistical analysis is indicated. Scale bar, 10 μm and (zoom) 1 μm. ( b ) WT and 20q-KO cells were analyzed for T-SCE events with G-rich (green) and C-rich (red) PNA probes. The fraction of chromosome ends with T-SCE obtained from three different experiments was quantified and graphed as the mean values±s.e.m., n =30 metaphases. The number of metaphases analyzed is shown. Only events in which interchange of both colours were quantified (see examples of no-T-SCE and T-SCE). The quantification was carried out by counting the number of events in the same chromosome or in different chromosomes and then normalizing it by the total number of chromosomes observed in each metaphase. Scale bar, 1 μm. ( c ) Quantification of <t>DNA-containing</t> double minute chromosomes (TDMs) in WT and 20q-KO cells from three different experiments (mean values±s.e.m., n =30 metaphases). An example of TDMs is shown. One-way Anova with Dunnett's post test was used for all statistical analysis (* P
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    Promega unidirectional exonuclease iii digestion
    Quantification of chromatin immunoprecipitation by real-time PCR. Chromatin from cross-linked subconfluent MCF7 or C33 cells was immunoprecipitated with antibodies specific for RB (C and D) and AP-2 (E and F). <t>bcl-2</t> or GAPDH sequences were detected by PCR analysis of eluted DNA using a LightCycler (Roche). A known amount of chromatin from each sample was removed before immunoprecipitation and PCR amplified like the immunoprecipitates (input, panels A and B). The curves show the accumulation of PCR products plotted against the number of cycles ( bcl-2 : panels A, C, and E; GAPDH: panels B, D, and F). The results are shown for only one dilution of the immunoprecipitates, but <t>three</t> dilutions were analyzed for each sample. Copy numbers were estimated by reference to a plasmid DNA containing the bcl-2 or GAPDH sequence using the second derivative maximum method. Curves obtained with reference plasmid DNA for bcl-2 (G) or GAPDH (H), using (from right to left in each case) 1, 10, 100, and 1,000 fg of plasmid, are shown (standard).
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    Image Search Results


    Deletion of the 20q-TERRA locus decreases telomere length and protection in U2OS cells. ( a ) Q-FISH images obtained from metaphases spreads from U2OS cells WT and KO for the Chr20q-TERRA locus (clones A4, B4 and C4). (Left graphs) Frequency graphs of telomere length (a.u.) distribution measured in WT and in the 20q-KO cells (clones A4, B4 and C4) from three independent experiments. The mean telomere length and the number of telomeres and metaphases analyzed is shown. The red lines are arbitrary lines placed in the exact same position in each frequency graph to visualize differences between the 20q-KO clones and the WT controls (right graphs) The mean telomere length, the percentage of short telomeres and the quantification of signal-free ends per metaphase are also represented. Short telomeres are considered those in the 10% percentile of the total telomere length distribution. Total number of metaphases used for the statistical analysis is indicated. Scale bar, 10 μm and (zoom) 1 μm. ( b ) WT and 20q-KO cells were analyzed for T-SCE events with G-rich (green) and C-rich (red) PNA probes. The fraction of chromosome ends with T-SCE obtained from three different experiments was quantified and graphed as the mean values±s.e.m., n =30 metaphases. The number of metaphases analyzed is shown. Only events in which interchange of both colours were quantified (see examples of no-T-SCE and T-SCE). The quantification was carried out by counting the number of events in the same chromosome or in different chromosomes and then normalizing it by the total number of chromosomes observed in each metaphase. Scale bar, 1 μm. ( c ) Quantification of DNA-containing double minute chromosomes (TDMs) in WT and 20q-KO cells from three different experiments (mean values±s.e.m., n =30 metaphases). An example of TDMs is shown. One-way Anova with Dunnett's post test was used for all statistical analysis (* P

    Journal: Nature Communications

    Article Title: Telomeric RNAs are essential to maintain telomeres

    doi: 10.1038/ncomms12534

    Figure Lengend Snippet: Deletion of the 20q-TERRA locus decreases telomere length and protection in U2OS cells. ( a ) Q-FISH images obtained from metaphases spreads from U2OS cells WT and KO for the Chr20q-TERRA locus (clones A4, B4 and C4). (Left graphs) Frequency graphs of telomere length (a.u.) distribution measured in WT and in the 20q-KO cells (clones A4, B4 and C4) from three independent experiments. The mean telomere length and the number of telomeres and metaphases analyzed is shown. The red lines are arbitrary lines placed in the exact same position in each frequency graph to visualize differences between the 20q-KO clones and the WT controls (right graphs) The mean telomere length, the percentage of short telomeres and the quantification of signal-free ends per metaphase are also represented. Short telomeres are considered those in the 10% percentile of the total telomere length distribution. Total number of metaphases used for the statistical analysis is indicated. Scale bar, 10 μm and (zoom) 1 μm. ( b ) WT and 20q-KO cells were analyzed for T-SCE events with G-rich (green) and C-rich (red) PNA probes. The fraction of chromosome ends with T-SCE obtained from three different experiments was quantified and graphed as the mean values±s.e.m., n =30 metaphases. The number of metaphases analyzed is shown. Only events in which interchange of both colours were quantified (see examples of no-T-SCE and T-SCE). The quantification was carried out by counting the number of events in the same chromosome or in different chromosomes and then normalizing it by the total number of chromosomes observed in each metaphase. Scale bar, 1 μm. ( c ) Quantification of DNA-containing double minute chromosomes (TDMs) in WT and 20q-KO cells from three different experiments (mean values±s.e.m., n =30 metaphases). An example of TDMs is shown. One-way Anova with Dunnett's post test was used for all statistical analysis (* P

    Article Snippet: The slides were treated with 0.5 mg ml−1 RNase A for 10 min at 37 °C, stained with 0.5 μg ml−1 Hoechst 33258 (Sigma) in 2 × SSC (0.3 M NaCl, 0.03 M sodium citrate) for 15 min at room temperature and then exposed to 365 nm UV light (Stratalinker 1800 UV irradiator) for 25–30 min. Enzymatic digestion of the BrdU/BrdC-substituted DNA strands with 3 U μl−1 of Exonuclease III (Promega) in buffer supplied by the manufacturer (50 mM Tris–HCl, 5 mM MgCl2 and 5 mM dithiothreitol, pH 8) was allowed to proceed for 10 min at room temperature.

    Techniques: Fluorescence In Situ Hybridization, Clone Assay

    Deletion of the 20q-TERRA locus decreases telomere protection in U2OS cells. ( a ) Quantification of the total γH2AX signal per nucleus (mean values±s.e.m., n =number of cells) is shown. The total number of cells analyzed is indicated. ( b ) Quantification of the total 53BP1 spot signal per nucleus (mean values±s.e.m., n =number of cells is shown). The total number of cells analyzed is indicated. ( c ) Graphs showing the quantification of the co-localization (TIF) between TRF2 and either γH2AX or 53BP1 in WT cells and in all 20q-KO clones (mean values±s.e.m., n =3 independent experiments for γH2AX and n =number of cells for 53BP1) per cell is shown. The total number of nuclei analyzed is indicated. ( d ) Representative images of the average number of TIFs found on double inmunostain to detect the telomere protein TRF2 (green) and either the DNA damage markers phospho-Histone γH2AX or 53BP1 (red) in the U2OS cells WT or deleted for the 20q locus. Arrowheads indicate co-localization events. Scale bar, 10 μm. ( e ) Quantification of chromosomal end-to-end fusions in WT and in the 20q-KO cells from three independent experiments (mean values±s.e.m., n =metaphases). Examples of end-to-end fusions are shown as well. Scale bar, 1 μm. ( f ) Array-CGH analysis was performed on hybridization on the same membrane of DNA differentially labelled from WT and 20q-KO cells. The chromosomal gains and losses in 20q-KO cells normalized by WT cells are represented. The chromosomal gains are shown in green and in red the chromosomal losses. One-way Anova with Dunnett's post test was used for all statistical analysis except for the quantification of chromosomal fusions in which the Student's t -test was used (* P

    Journal: Nature Communications

    Article Title: Telomeric RNAs are essential to maintain telomeres

    doi: 10.1038/ncomms12534

    Figure Lengend Snippet: Deletion of the 20q-TERRA locus decreases telomere protection in U2OS cells. ( a ) Quantification of the total γH2AX signal per nucleus (mean values±s.e.m., n =number of cells) is shown. The total number of cells analyzed is indicated. ( b ) Quantification of the total 53BP1 spot signal per nucleus (mean values±s.e.m., n =number of cells is shown). The total number of cells analyzed is indicated. ( c ) Graphs showing the quantification of the co-localization (TIF) between TRF2 and either γH2AX or 53BP1 in WT cells and in all 20q-KO clones (mean values±s.e.m., n =3 independent experiments for γH2AX and n =number of cells for 53BP1) per cell is shown. The total number of nuclei analyzed is indicated. ( d ) Representative images of the average number of TIFs found on double inmunostain to detect the telomere protein TRF2 (green) and either the DNA damage markers phospho-Histone γH2AX or 53BP1 (red) in the U2OS cells WT or deleted for the 20q locus. Arrowheads indicate co-localization events. Scale bar, 10 μm. ( e ) Quantification of chromosomal end-to-end fusions in WT and in the 20q-KO cells from three independent experiments (mean values±s.e.m., n =metaphases). Examples of end-to-end fusions are shown as well. Scale bar, 1 μm. ( f ) Array-CGH analysis was performed on hybridization on the same membrane of DNA differentially labelled from WT and 20q-KO cells. The chromosomal gains and losses in 20q-KO cells normalized by WT cells are represented. The chromosomal gains are shown in green and in red the chromosomal losses. One-way Anova with Dunnett's post test was used for all statistical analysis except for the quantification of chromosomal fusions in which the Student's t -test was used (* P

    Article Snippet: The slides were treated with 0.5 mg ml−1 RNase A for 10 min at 37 °C, stained with 0.5 μg ml−1 Hoechst 33258 (Sigma) in 2 × SSC (0.3 M NaCl, 0.03 M sodium citrate) for 15 min at room temperature and then exposed to 365 nm UV light (Stratalinker 1800 UV irradiator) for 25–30 min. Enzymatic digestion of the BrdU/BrdC-substituted DNA strands with 3 U μl−1 of Exonuclease III (Promega) in buffer supplied by the manufacturer (50 mM Tris–HCl, 5 mM MgCl2 and 5 mM dithiothreitol, pH 8) was allowed to proceed for 10 min at room temperature.

    Techniques: Clone Assay, Hybridization

    Schema of ReDFISH technique. ( a ) ReDFISH of a chromosome that has replicated fully in the presence of BrdUrd/dC. Newly synthesized DNA incorporating BrdUrd/dC (horizontal stripes) is removed after nicking the DNA with Hoechst 33258 plus UV and digesting nicked DNA with exonuclease III, leaving only the parental strands. The G-rich telomeric strand is the template for lagging strand synthesis and anneals to a Cy3-conjugated C-rich telomeric probe, whereas the C-rich telomeric strand is the template for leading strand synthesis and anneals to an FITC-conjugated G-rich telomeric probe. This pattern defines which telomeric strands replicated at the time of BrdUrd/BrdC labeling. ( b ) ReDFISH of a partially replicated chromosome. In this example, only the p-arm telomere of the X chromosome was replicating during the 1-h BrdUrd/dC pulse; the q arm was not replicating. As a consequence, only the parental strands are available for hybridization in the p arm (schema, p arms), whereas both strands of unreplicated q-arm DNA survive digestion and hybridize to both probes (schema, q arms).

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Asynchronous replication timing of telomeres at opposite arms of mammalian chromosomes

    doi: 10.1073/pnas.0404106101

    Figure Lengend Snippet: Schema of ReDFISH technique. ( a ) ReDFISH of a chromosome that has replicated fully in the presence of BrdUrd/dC. Newly synthesized DNA incorporating BrdUrd/dC (horizontal stripes) is removed after nicking the DNA with Hoechst 33258 plus UV and digesting nicked DNA with exonuclease III, leaving only the parental strands. The G-rich telomeric strand is the template for lagging strand synthesis and anneals to a Cy3-conjugated C-rich telomeric probe, whereas the C-rich telomeric strand is the template for leading strand synthesis and anneals to an FITC-conjugated G-rich telomeric probe. This pattern defines which telomeric strands replicated at the time of BrdUrd/BrdC labeling. ( b ) ReDFISH of a partially replicated chromosome. In this example, only the p-arm telomere of the X chromosome was replicating during the 1-h BrdUrd/dC pulse; the q arm was not replicating. As a consequence, only the parental strands are available for hybridization in the p arm (schema, p arms), whereas both strands of unreplicated q-arm DNA survive digestion and hybridize to both probes (schema, q arms).

    Article Snippet: The nicked BrdUrd/dC-substituted DNA was digested with 3 units/μl exonuclease III (Promega) in 50 mM Tris·HCl (pH 8.0), 5 mM MgCl2 , and 5 mM DTT for 10 min at room temperature.

    Techniques: Synthesized, Labeling, Hybridization

    Secondary structure of satRPV RNA monomer. (A) Autoradiograph of 5′-end-labeled transcript of monomeric satRPV RNA after partial digestion with imidazole or RNase T 1 . Gel-purified, end-labeled RNA was incubated in three different molarities of imidazole under nondenaturing (native) conditions for 15 h (lanes 3 to 6). Indicated units of RNase T 1 were used for digestion in nondenaturing (native) conditions for 5 min at 25°C. To generate the G-track sequencing ladder, indicated units of RNase T 1 ) superimposed with markers indicating intensity of cleavages in panel A. Open, filled, and double symbols indicate weak, moderate, and strong cuts, respectively.

    Journal: Journal of Virology

    Article Title: cis and trans Requirements for Rolling Circle Replication of a Satellite RNA

    doi: 10.1128/JVI.78.6.3072-3082.2004

    Figure Lengend Snippet: Secondary structure of satRPV RNA monomer. (A) Autoradiograph of 5′-end-labeled transcript of monomeric satRPV RNA after partial digestion with imidazole or RNase T 1 . Gel-purified, end-labeled RNA was incubated in three different molarities of imidazole under nondenaturing (native) conditions for 15 h (lanes 3 to 6). Indicated units of RNase T 1 were used for digestion in nondenaturing (native) conditions for 5 min at 25°C. To generate the G-track sequencing ladder, indicated units of RNase T 1 ) superimposed with markers indicating intensity of cleavages in panel A. Open, filled, and double symbols indicate weak, moderate, and strong cuts, respectively.

    Article Snippet: Serial deletion mutants (see Fig. ) of satRPV were generated by using exonuclease III (ExoIII; Erase-a-Base system; Promega) after digestion of plasmid pWT with Msc I (construct M122), Ava I (A222, A241, A243) or Cla I (C321).

    Techniques: Autoradiography, Labeling, Purification, Incubation, Sequencing

    Quantification of chromatin immunoprecipitation by real-time PCR. Chromatin from cross-linked subconfluent MCF7 or C33 cells was immunoprecipitated with antibodies specific for RB (C and D) and AP-2 (E and F). bcl-2 or GAPDH sequences were detected by PCR analysis of eluted DNA using a LightCycler (Roche). A known amount of chromatin from each sample was removed before immunoprecipitation and PCR amplified like the immunoprecipitates (input, panels A and B). The curves show the accumulation of PCR products plotted against the number of cycles ( bcl-2 : panels A, C, and E; GAPDH: panels B, D, and F). The results are shown for only one dilution of the immunoprecipitates, but three dilutions were analyzed for each sample. Copy numbers were estimated by reference to a plasmid DNA containing the bcl-2 or GAPDH sequence using the second derivative maximum method. Curves obtained with reference plasmid DNA for bcl-2 (G) or GAPDH (H), using (from right to left in each case) 1, 10, 100, and 1,000 fg of plasmid, are shown (standard).

    Journal: Molecular and Cellular Biology

    Article Title: The Retinoblastoma Protein Binds the Promoter of the Survival Gene bcl-2 and Regulates Its Transcription in Epithelial Cells through Transcription Factor AP-2

    doi: 10.1128/MCB.22.22.7877-7888.2002

    Figure Lengend Snippet: Quantification of chromatin immunoprecipitation by real-time PCR. Chromatin from cross-linked subconfluent MCF7 or C33 cells was immunoprecipitated with antibodies specific for RB (C and D) and AP-2 (E and F). bcl-2 or GAPDH sequences were detected by PCR analysis of eluted DNA using a LightCycler (Roche). A known amount of chromatin from each sample was removed before immunoprecipitation and PCR amplified like the immunoprecipitates (input, panels A and B). The curves show the accumulation of PCR products plotted against the number of cycles ( bcl-2 : panels A, C, and E; GAPDH: panels B, D, and F). The results are shown for only one dilution of the immunoprecipitates, but three dilutions were analyzed for each sample. Copy numbers were estimated by reference to a plasmid DNA containing the bcl-2 or GAPDH sequence using the second derivative maximum method. Curves obtained with reference plasmid DNA for bcl-2 (G) or GAPDH (H), using (from right to left in each case) 1, 10, 100, and 1,000 fg of plasmid, are shown (standard).

    Article Snippet: The Bcl-2 CAT −1150, −1093, −946, −744, −670, −656, −648, −447, −231 and −134 constructs were constructed from Bcl-2P1P2CAT by unidirectional exonuclease III digestion of the bcl-2 promoter with the Erase-a-Base kit (Promega).

    Techniques: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Immunoprecipitation, Polymerase Chain Reaction, Amplification, Plasmid Preparation, Sequencing

    Cell-type-specific activation of bcl-2 promoter by RB. (A) Effects of RB on the activity of Bcl-2 P1P2 and Bcl-2 P1 promoters in MDCK epithelial cells. One microgram of each Bcl-2 CAT construct was transfected with 5 μg of pSV-RB expression vector (black bars) or the pSV-RBΔ vector as a control (white bars). The results shown are averages of values obtained in three independent experiments expressed as fold activation of CAT activity relative to the basal promoter activity, which is assigned a value of 1. Error bars indicate standard deviation. (B) Dose-dependent effect of RB on transcriptional activity of the Bcl-2 P1P2 promoter in MDCK epithelial cells and in NIH 3T3 fibroblasts. One microgram of Bcl-2 P1P2 CAT construct was cotransfected with increasing amounts of pSV-RB expression vector (solid lines, filled circles) or the pSV-RBΔ vector as a control (stippled lines, triangles). Each value is expressed as fold activation of CAT activity relative to the baseline value obtained by cotransfecting the Bcl-2 P1P2 CAT vector with the empty expression vector. The results shown are the averages of values obtained in three independent experiments performed in duplicate. Error bars indicate standard deviations. (C) Effects of RB on the activity of Bcl-2 P2 (−1093) promoter in MDCK epithelial cells, in two stably transformed cell lines: fibroblast-like MDCK(1-6) transformed by wild-type LT and epithelial-cell-like MDCK(2a5) transformed by mutated LT(K1) and in MCF7 cells. Cells were transfected with 1 μg of the Bcl-2 P2 (−1093) CAT construct and 5 μg of the pSV-RB expression vector (black bars) or the empty expression vector (white bars). The results are averages of values obtained in three independent experiments performed in duplicate and are expressed as in panel A.

    Journal: Molecular and Cellular Biology

    Article Title: The Retinoblastoma Protein Binds the Promoter of the Survival Gene bcl-2 and Regulates Its Transcription in Epithelial Cells through Transcription Factor AP-2

    doi: 10.1128/MCB.22.22.7877-7888.2002

    Figure Lengend Snippet: Cell-type-specific activation of bcl-2 promoter by RB. (A) Effects of RB on the activity of Bcl-2 P1P2 and Bcl-2 P1 promoters in MDCK epithelial cells. One microgram of each Bcl-2 CAT construct was transfected with 5 μg of pSV-RB expression vector (black bars) or the pSV-RBΔ vector as a control (white bars). The results shown are averages of values obtained in three independent experiments expressed as fold activation of CAT activity relative to the basal promoter activity, which is assigned a value of 1. Error bars indicate standard deviation. (B) Dose-dependent effect of RB on transcriptional activity of the Bcl-2 P1P2 promoter in MDCK epithelial cells and in NIH 3T3 fibroblasts. One microgram of Bcl-2 P1P2 CAT construct was cotransfected with increasing amounts of pSV-RB expression vector (solid lines, filled circles) or the pSV-RBΔ vector as a control (stippled lines, triangles). Each value is expressed as fold activation of CAT activity relative to the baseline value obtained by cotransfecting the Bcl-2 P1P2 CAT vector with the empty expression vector. The results shown are the averages of values obtained in three independent experiments performed in duplicate. Error bars indicate standard deviations. (C) Effects of RB on the activity of Bcl-2 P2 (−1093) promoter in MDCK epithelial cells, in two stably transformed cell lines: fibroblast-like MDCK(1-6) transformed by wild-type LT and epithelial-cell-like MDCK(2a5) transformed by mutated LT(K1) and in MCF7 cells. Cells were transfected with 1 μg of the Bcl-2 P2 (−1093) CAT construct and 5 μg of the pSV-RB expression vector (black bars) or the empty expression vector (white bars). The results are averages of values obtained in three independent experiments performed in duplicate and are expressed as in panel A.

    Article Snippet: The Bcl-2 CAT −1150, −1093, −946, −744, −670, −656, −648, −447, −231 and −134 constructs were constructed from Bcl-2P1P2CAT by unidirectional exonuclease III digestion of the bcl-2 promoter with the Erase-a-Base kit (Promega).

    Techniques: Activation Assay, Activity Assay, Construct, Transfection, Expressing, Plasmid Preparation, Standard Deviation, Stable Transfection, Transformation Assay

    Identification of the region of the bcl-2 promoter that responds to RB protein in MDCK epithelial cells. (A) MDCK cells were transfected with a series of bcl-2 promoter deletion constructs (1 μg of each) and either 5 μg of pSV-RB expression vector (black bars) or the empty expression vector (white bars). The results are averages of values obtained in three independent experiments and are expressed as promoter activity relative to the basal promoter activity, which is assigned a value of 1. Error bars indicate standard deviations. bcl-2 constructs are schematically represented on the left. Numbering of the bcl-2 sequence is relative to the translation start site. (B) Fine mapping of the RB response element. One microgram of each bcl-2 construct (with the 5′ extremity deleted as indicated) was transfected with 5 μg of pSV-RB expression vector (black bars) or the empty expression vector (white bars). Experiments were performed and results were expressed as in panel A. (C) MCF7 cells were transfected as in panel A with three bcl-2 promoter constructs.

    Journal: Molecular and Cellular Biology

    Article Title: The Retinoblastoma Protein Binds the Promoter of the Survival Gene bcl-2 and Regulates Its Transcription in Epithelial Cells through Transcription Factor AP-2

    doi: 10.1128/MCB.22.22.7877-7888.2002

    Figure Lengend Snippet: Identification of the region of the bcl-2 promoter that responds to RB protein in MDCK epithelial cells. (A) MDCK cells were transfected with a series of bcl-2 promoter deletion constructs (1 μg of each) and either 5 μg of pSV-RB expression vector (black bars) or the empty expression vector (white bars). The results are averages of values obtained in three independent experiments and are expressed as promoter activity relative to the basal promoter activity, which is assigned a value of 1. Error bars indicate standard deviations. bcl-2 constructs are schematically represented on the left. Numbering of the bcl-2 sequence is relative to the translation start site. (B) Fine mapping of the RB response element. One microgram of each bcl-2 construct (with the 5′ extremity deleted as indicated) was transfected with 5 μg of pSV-RB expression vector (black bars) or the empty expression vector (white bars). Experiments were performed and results were expressed as in panel A. (C) MCF7 cells were transfected as in panel A with three bcl-2 promoter constructs.

    Article Snippet: The Bcl-2 CAT −1150, −1093, −946, −744, −670, −656, −648, −447, −231 and −134 constructs were constructed from Bcl-2P1P2CAT by unidirectional exonuclease III digestion of the bcl-2 promoter with the Erase-a-Base kit (Promega).

    Techniques: Transfection, Construct, Expressing, Plasmid Preparation, Activity Assay, Sequencing