Journal: Molecular and Cellular Biology

Article Title: TIN2-Tethered TPP1 Recruits Human Telomerase to Telomeres In Vivo

doi: 10.1128/mcb.00240-10

Figure Lengend Snippet: FIG. 6. The TPP1 OB-fold is required to rescue telomerase recruitment to telomeres. An shRNA-resistant form of TPP1 is able to restore hTR localization to telomeres in TPP1-depleted cells. However, an shRNA-resistant form of TPP1 lacking the OB-fold cannot restore localization. (A) Parental and TPP1-depleted super-telomerase HeLa cells were subjected to FISH and IF to detect hTR (red), coilin (blue), and TRF2 (green). Merge panels show superimposition of hTR, coilin, and TRF2. Next, parental cells were cotransfected with shTPP1 and either TPP1* or TPP1OB*. Treated cells were subjected to FISH and IF to detect hTR (red), FLAG (blue), and RAP1 (telomere marker, green). Merge panels show superimposition of hTR, FLAG, and RAP1. (B) Plot of the average number of telomere-associated hTR foci per cell in the parental cells and each experimental group. Error bars indicate standard errors calculated with N equal to the number of samples quantitated.

Article Snippet: Next, cells were incubated with one of several combinations of the following primary antibodies at the indicated dilution for 1 h at room temperature: mouse anti-p80 coilin (1:5,000, ) (1), mouse anti-TRF2 (1:1,000; Imgenex Corp., San Diego, CA), rabbit anti-hTERT (1:400; Rockland, Gilbertsville, PA), mouse anti-FLAG (1:500; Sigma-Aldrich, St. Louis, MO), rabbit anti-RAP1(1: 2,000; Novus Biologicals, Littleton, CO), and rabbit anti-53BP1(1:500; Bethyl, Montgomery, TX).

Techniques: shRNA, Marker

Journal: Journal of Cell Science

Article Title: Talin, a Rap1 effector for integrin activation at the plasma membrane, also promotes Rap1 activity by disrupting sequestration of Rap1 by SHANK3

doi: 10.1242/jcs.263595

Figure Lengend Snippet: Optogenetic recruitment of talin to the plasma membrane of endothelial cells leads to activation of Rap1. (A) Schematic representation of the optogenetic constructs CIBN–GFP–CAAX and CRY2–mCherry–talin expressed in immortalized mouse lung endothelial cells. The CIBN moiety is anchored to the plasma membrane and recruits CRY2–mCherry–talin upon exposure of the cells to 450 nm (blue) light. Previous work has shown that such recruitment leads to activation of integrin αVβ3 . Rap1 activation, the transition from Rap1–GDP to Rap1–GTP, can also be monitored during this process. (B) Time course of Rap1 activation in endothelial cells in response to blue light illumination. Rap1–GTP was selectively pulled down using agarose beads loaded with the Rap-binding domain of RalGDS and detected using an anti-Rap1 antibody. Upper panel: representative western blots of the Rap1–GTP pull-down assay and total Rap1 in whole-cell lysates (input: 5%). Lower panel: quantitative analysis of Rap1–GTP. The ratio of Rap1–GTP to total Rap1 was calculated and normalized to that observed at time zero when the cells were maintained in the dark. Data represent means±s.e.m. of four experiments (* P <0.05; paired two-tailed Student's t -test). (C) Time course of Rap1 activation in response to blue light in A5 CHO cells stably expressing integrin αIIbβ3, CIBN–GFP–CAAX and CRY2–mCherry–talin. Data represent means±s.e.m. of four experiments (* P <0.05; ** P <0.01; paired two-tailed Student's t -test). (D) Recruitment to the plasma membrane of a CRY2–mCherry–talin mutant (R118E) that cannot interact with Rap1 fails to activate Rap1 in A5 CHO cells. Data represent means±s.e.m. of four experiments (N.S., not significant; ** P <0.01; paired two-tailed Student's t -test). (E) Expression of a CRY2–mCherry–talin mutant (L325R) defective in activating integrins still enables Rap1 activation in these cells upon recruitment of CRY2–mCherry–talin to the plasma membrane in A5 CHO cells. Data represent means±s.e.m. of four experiments (* P <0.05; paired two-tailed Student's t -test).

Article Snippet: The mouse monoclonal antibody to Rap1 was from Proteintech (Rosemont, IL, USA; 67174).

Techniques: Clinical Proteomics, Membrane, Activation Assay, Construct, Binding Assay, Western Blot, Pull Down Assay, Two Tailed Test, Stable Transfection, Expressing, Mutagenesis

Journal: Journal of Cell Science

Article Title: Talin, a Rap1 effector for integrin activation at the plasma membrane, also promotes Rap1 activity by disrupting sequestration of Rap1 by SHANK3

doi: 10.1242/jcs.263595

Figure Lengend Snippet: Optogenetic recruitment of talin to the plasma membrane promotes active Rap1 localization to cell edges. (A) Optogenetic recruitment of talin to the plasma membrane promotes active Rap1 localization to the cell periphery in suspended cells. Immortalized murine endothelial cells in suspension expressing CIBN–GFP–CAAX and CRY2–mCherry–talin were illuminated using blue light for 30 min before Rap1–GTP was detected in situ as described in the Materials and Methods. Samples without GST–RalGDS were used as a control (column 1). White arrows indicate enrichment of active Rap1 and CRY2–mCherry–talin at the cell periphery, the former only in response to blue light. Scale bar: 10 µm. (B) Optogenetic recruitment of talin to the plasma membrane enriches active Rap1 localization at cell protrusions in adherent cells. Immortalized murine endothelial cells expressing CIBN–GFP–CAAX and CRY2–mCherry–talin were plated on fibronectin-coated coverslips for 30 min before being illuminated with blue light or maintained in the dark for 30 min. Samples were fixed and in situ Rap1–GTP assay was performed as described in the Materials and Methods. Samples without GST–RalGDS incubation were used as a negative control and are shown in column 1. In these representative images, cell protrusions are highlighted by the small box in the main panel and presented as magnified insets on the bottom right of each image. Blue light illumination induces active Rap1 localization on cell lamellipodium-like protrusions (column 4) and pseudopodium-like protrusions (column 5). Note that such signal enrichment was not seen in cells maintained in the dark (columns 2 and 3) despite the formation of cell protrusions. Scale bars: 20 µm (main panel); 1 µm (inset). Images in A,B are representative of three independent experiments.

Article Snippet: The mouse monoclonal antibody to Rap1 was from Proteintech (Rosemont, IL, USA; 67174).

Techniques: Clinical Proteomics, Membrane, Suspension, Expressing, In Situ, Control, Incubation, Negative Control

Journal: Journal of Cell Science

Article Title: Talin, a Rap1 effector for integrin activation at the plasma membrane, also promotes Rap1 activity by disrupting sequestration of Rap1 by SHANK3

doi: 10.1242/jcs.263595

Figure Lengend Snippet: Overexpression of SHANK3 blocks Rap1 activation induced by talin recruitment to the plasma membrane. (A–C) SHANK3 tagged with Myc–mAzurite was transfected into A5 CHO cells stably expressing CIBN–GFP–CAAX and CRY2–mCherry–talin. Cells triple positive for mAzurite, GFP and mCherry were sorted by flow cytometry. Cells expressing Myc–mAzurite without SHANK3 served as a control. (A) Western blot analysis of SHANK3–Myc–mAzurite expression in these cells. β-actin served as a loading control. SHANK3 overexpression did not affect the levels of CRY2–mCherry–talin. The images represent two independent experiments. (B) SHANK3 overexpression inhibits Rap1 activation in response to the optogenetic recruitment of talin to the plasma membrane. Data represent means±s.e.m. of five experiments (N.S., not significant; ** P <0.01; paired two-tailed Student's t -test). (C) SHANK3 blunts activation of integrin αIIbβ3 in response to the optogenetic recruitment of talin to the plasma membrane. Activation of integrin αIIbβ3 was monitored by flow cytometry using the PAC-1 antibody and expressed as the fold increase relative to that observed when cells were maintained in the dark. Data represent means±s.e.m. of five experiments (* P <0.05; paired two-tailed Student's t -test). (D–G) Lentiviruses encoding the WT SPN domain of SHANK3 [mAzurite–FLAG–SPN (WT)], the R12C SPN mutant [mAzurite–FLAG–SPN (R12C)] or the L68P SPN mutant [Myc–mAzurite–FLAG–SPN (L68P)] were transduced into immortalized murine lung endothelial cells expressing CIBN–GFP–CAAX and CRY2–mCherry–talin. Cells infected with empty lentiviral vector served as controls. (D) WT SPN, but not R12C or L68P SPN, inhibits Rap1 activation following optogenetic recruitment of talin to the plasma membrane. Data represent mean±s.e.m. of four experiments (N.S., not significant; * P <0.05; paired two-tailed Student's t -test). (E) WT SPN, but not the R12C or L68P SPN mutants, inhibits specific fibrinogen binding to integrin αVβ3 upon optogenetic recruitment of talin to the plasma membrane. Data represent means±s.e.m. of eight experiments (N.S., not significant; * P <0.05; ** P <0.01; paired two-tailed Student's t -test). (F,G) Duolink proximity ligation assay (PLA) was performed to examine the effects of SHANK3 SPN on the association of Rap1 with CRY2–mCherry–talin in endothelial cells. (F) Schematic representation of the Duolink PLA. Created in BioRender by Liao, Z., 2025. https://BioRender.com/m47c469 . This figure was sublicensed under CC-BY 4.0 terms. (G) After 30 min of incubation at room temperature in the absence or presence of blue light illumination, cells were fixed, permeabilized and stained with rabbit anti-mCherry and mouse anti-Rap1 antibodies. Then, Duolink PLA flow cytometry was performed to assess the interaction between CRY2–mCherry–talin and Rap1. Cells kept in the dark and untreated with primary antibodies served as controls. Data represent means±s.e.m. of four experiments (* P <0.05; paired two-tailed Student's t -test).

Article Snippet: The mouse monoclonal antibody to Rap1 was from Proteintech (Rosemont, IL, USA; 67174).

Techniques: Over Expression, Activation Assay, Clinical Proteomics, Membrane, Transfection, Stable Transfection, Expressing, Flow Cytometry, Control, Western Blot, Two Tailed Test, Mutagenesis, Infection, Plasmid Preparation, Binding Assay, Proximity Ligation Assay, Incubation, Staining

Journal: Journal of Cell Science

Article Title: Talin, a Rap1 effector for integrin activation at the plasma membrane, also promotes Rap1 activity by disrupting sequestration of Rap1 by SHANK3

doi: 10.1242/jcs.263595

Figure Lengend Snippet: Optogenetic recruitment of talin to the plasma membrane impairs Rap1 interaction with SHANK3. (A) Schematic representation of the Duolink proximity ligation assay (PLA). Cells were fixed, permeabilized and stained with rabbit anti-SHANK3 and mouse anti-Rap1 antibodies before Duolink PLA was performed to assess the proximity of SHANK3 to Rap1. Created in BioRender by Liao, Z., 2025. https://BioRender.com/b40n281 . This figure was sublicensed under CC-BY 4.0 terms. (B) Immortalized murine lung endothelial cells expressing CRY2–mCherry–talin and CIBN–GFP–CAAX were plated on fibrinogen overnight, fixed, permeabilized and stained with anti-SHANK3 and anti-Rap1 antibodies. PLA was performed to evaluate colocalization of endogenous SHANK3 and Rap1. Cell nuclei were counterstained with DAPI and cells were imaged by confocal microscopy. Cells kept in the dark and untreated with primary antibodies served as controls. Two areas within the images with merged signals for PLA and CIBN–GFP–CAAX are highlighted with boxes and presented as magnified insets on the bottom. PLA signals were observed within the cytoplasm (inset on the left) and on the plasma membrane (inset on the right). Scale bars: 35 µm (main panel); 10 µm (inset). Images represent two independent experiments. (C) Immortalized murine lung endothelial cells in suspension were either kept in the dark or exposed to blue light illumination for the indicated times, before being subjected to Duolink PLA assay and analyzed by flow cytometry to quantitatively assess the interaction of endogenous SHANK3 and Rap1. Cells kept in the dark and untreated with primary antibodies served as controls. Data represent means±s.e.m. of five experiments (N.S., not significant; * P <0.05; paired two-tailed Student's t -test). Cells transduced with lentivirus encoding shRNA to knock down SHANK3 were also used as a further control to demonstrate the specificity of the PLA signal in four out of the five experiments (* P <0.05; unpaired two-tailed Student's t -test).

Article Snippet: The mouse monoclonal antibody to Rap1 was from Proteintech (Rosemont, IL, USA; 67174).

Techniques: Clinical Proteomics, Membrane, Proximity Ligation Assay, Staining, Expressing, Confocal Microscopy, Suspension, Flow Cytometry, Two Tailed Test, Transduction, shRNA, Knockdown, Control

Journal: Science signaling

Article Title: Mechanisms of autoregulation of C3G, activator of the GTPase Rap1, and its catalytic deregulation in lymphomas.

doi: 10.1126/scisignal.abb7075

Figure Lengend Snippet: Fig. 4. Mutations that disrupt the AIR/Cdc25HD interaction activate C3G constitutively. (A) Conservation scores of C3G per residue (colored bars) and average values per domain (boxes). Alignment of the sequences of the P3 motif and part of the AIR from representative species; positions are colored according to their conservation. Secondary structure predictions by three methods are shown under the sequences. Secondary structure prediction for the com- plete SH3b domain is shown in fig. S2. (B and C) Binding of the Cdc25HD to GST-AIR, WT, and mutants, analyzed by PD assays. Cdc25HD in the PD was detected by Western blot. Two types of substitutions were assayed: reverse-charge replacements (B) and changes to alanine and mutations described in lymphomas (C). PDs are representative of two independent experiments. (D) Helical wheel representation of the predicted helix in the AIR-CBR. Met551, Tyr554, and Met555 define the putative binding site for the Cdc25HD. (E and F) Representative nucleotide exchange reactions of Rap1:mant-dGDP catalyzed by full-length C3G WT and the indicated point mutants (1 M) (E) and the exchange rates (kobs) (F). n = 3 to 5 independent experiments, as indicated, one shown in (E). (G) Schematic representation of auto inhibited C3G and the uninhibited conformation induced by mutations that destabilize the AIR/Cdc25HD interaction.

Article Snippet: 13, eabb7075 (2020) 1 September 2020 13 of 17 rabbit pAb 121 against Rap1 (sc-65, Santa Cruz Biotechnology), and mouse mAb against -actin (dilution 1/2000; AC-15, SigmaAldrich).

Techniques: Residue, Binding Assay, Western Blot

Journal: Science signaling

Article Title: Mechanisms of autoregulation of C3G, activator of the GTPase Rap1, and its catalytic deregulation in lymphomas.

doi: 10.1126/scisignal.abb7075

Figure Lengend Snippet: Fig. 5. Lymphoma-related mutations in C3G activate Rap1 and LFA-1 in cells. (A) Analysis of Rap1-GTP in HEK293T cells expressing mEGFP; C3G-mEGFP WT; the mutants M551R, Y554H, or M555K; or the membrane-targeted WT with a CAAX sequence. Expression of mEGFP and endogenous Rap1 and -actin in cell lysates were analyzed by Western blot. In the PD, Rap1-GTP was detected by Western blot, and GST-RalGDS-RBD was detected by Ponceau S staining. PD is representative of three independent experiments. (B) Scatterplot and bar chart of three independent measurements of Rap1 activation in HEK293T cells, as described and represented in (A). Rap1-GTP levels in cells expressing C3G-mEGFP-CAAX were used to normalize the data from different experiments. (C) Correlation between the exchange activity in vitro (kobs) of C3G WT and mutants (shown in Fig. 4F) and the Rap1-GTP levels that they induced in HEK293T cells [shown in (B)]. (D) Rap1-GTP levels in Ba/F3 cells expressing C3G-mEGFP, WT or the indicated mutants, or mEGFP alone and in uninfected cells. Proteins were detected in the cell lysates and in the GST-RalGDS- RBD PDs as described in (A). In addition, C3G (endogenous and mEGFP-tagged) was also detected using an antibody against C3G. PD is representative of two independent experiments. (E) Time course activation of Rap1 after stimulation with IL-3 of Ba/F3 cells expressing mEGFP, C3G-mEGFP WT, or Y554H. Rap1-GTP was detected as described in (A) and (D). PD is representative of two independent experiments. (F) Activation of the integrin LFA-1 in Ba/F3 cells expressing mEGFP, C3G-mEGFP WT, or mutants, determined by flow cytometry (n = 3, biological replicates, means ± SD). Statistical comparison to cells expressing mEGFP was analyzed using ANOVA followed by Dunnett’s test. ***P < 0.001; ****P < 0.0001.

Article Snippet: 13, eabb7075 (2020) 1 September 2020 13 of 17 rabbit pAb 121 against Rap1 (sc-65, Santa Cruz Biotechnology), and mouse mAb against -actin (dilution 1/2000; AC-15, SigmaAldrich).

Techniques: Expressing, Membrane, Sequencing, Western Blot, Staining, Activation Assay, Activity Assay, In Vitro, Flow Cytometry, Comparison

Journal: Science signaling

Article Title: Mechanisms of autoregulation of C3G, activator of the GTPase Rap1, and its catalytic deregulation in lymphomas.

doi: 10.1126/scisignal.abb7075

Figure Lengend Snippet: Fig. 6. The AIR/Cdc25HD interaction is the main autoinhibitory mechanism and is disrupted by CrkL for activation. (A) Phosphorylation of purified C3G-WT with SrcKD, analyzed by Western blot. Similar analysis of Cdc25HD and the C3G mutants M551R, Y554R, and Y554H are shown in fig. S3 (A to D). (B) Representative dissociation reactions of Rap1:mant-dGDP catalyzed by C3G (1 M), unmodified or phosphorylated with SrcKD (p-C3G), alone and in the presence of CrkL (10 M). (C) Nucleotide ex- change rates (kobs) catalyzed by full-length C3G WT and point mutants and the isolated Cdc25HD. Unphosphorylated and SrcKD-phosphorylated samples were analyzed alone and in the presence of CrkL, n = 3 to 10 independent experiments as indicated. Representative dissociation reactions are shown in (B) (WT) and in fig. S3 (A to D) (Cdc25HD and mutants). (D) Analysis by PD of the competition of CrkL with the Cdc25HD for binding to four constructs of the AIR that contain the P3 and P4 (GST-537- 646-WT), only the P3 (GST-537-646-P4A), only the P4 (GST-545-646-WT), or none of these proline-rich motifs (GST-545-646-P4A). PDs are representative of two independent experiments. (E) Time course of the in vitro phosphorylation of the AIR (537-646) by the SrcKD, analyzed by Western blot; representative of two independent experiments. (F) Binding of Cdc25HD to GST-AIR (537-646) phosphorylated with SrcKD in PD assays. PD is representative of two independent experiments. (G) Exchange activity (kobs) of the Cdc25HD alone and in the presence of AIR (untagged or GST-fusion) or the AIR mutants in (D). n = 3 to 4 independent experiments as indicated. Representative dissociation experiments are shown in fig. S3E. (H) Schematic representation of the effect of phosphorylation and CrkL binding to the AIR on the release of the inhibitory interaction when the AIR and the Cdc25HD are assayed as individual proteins.

Article Snippet: 13, eabb7075 (2020) 1 September 2020 13 of 17 rabbit pAb 121 against Rap1 (sc-65, Santa Cruz Biotechnology), and mouse mAb against -actin (dilution 1/2000; AC-15, SigmaAldrich).

Techniques: Activation Assay, Phospho-proteomics, Purification, Western Blot, Isolation, Binding Assay, Construct, In Vitro, Activity Assay

Journal: Science signaling

Article Title: Mechanisms of autoregulation of C3G, activator of the GTPase Rap1, and its catalytic deregulation in lymphomas.

doi: 10.1126/scisignal.abb7075

Figure Lengend Snippet: Fig. 7. The NTD/REM interaction stimulates the GEF activity of the Cdc25HD. (A) Schematic representation of full-length C3G-WT, truncation fragments, and the mutant E731R/E784R (red crosses) in which the NTD/REM interaction is destabilized. (B to E) Nucleotide exchange activity (kobs) of the proteins depicted in (A), in the absence of stimuli (B), in the presence of CrkL (C), phosphorylated with SrcKD (D), and when CrkL and phosphorylation were combined (E). The number of independent experiments (n) is indicated. Lines are means ± SD. Representative nucleotide dissociation reactions are shown in fig. S4 (A, B, D, and E). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 by one-way ANOVA followed by Dunnett’s test (B and C) or unequal variance Brown-Forsythe ANOVA followed by Dunnett’s test (D and E). (F) Analysis of Rap1 activation in HEK293T cells expressing C3G-mEGFP WT, the mutant E731R/E784R, or C3G with the membrane targeting CAAX-tag. Results of two independent experiments are shown. (G) Schematic illustration of the contribution of the NTD/REM interaction to the activation by CrkL and phosphorylation of C3G WT and mutants.

Article Snippet: 13, eabb7075 (2020) 1 September 2020 13 of 17 rabbit pAb 121 against Rap1 (sc-65, Santa Cruz Biotechnology), and mouse mAb against -actin (dilution 1/2000; AC-15, SigmaAldrich).

Techniques: Activity Assay, Mutagenesis, Phospho-proteomics, Activation Assay, Expressing, Membrane

Journal: Science Advances

Article Title: TERRA transcripts localize at long telomeres to regulate telomerase access to chromosome ends

doi: 10.1126/sciadv.adk4387

Figure Lengend Snippet: ( A ) Detection of TERRA and hTR by smiFISH in HeLa cells. The percentage of cells, in which at least 1 TERRA-hTR colocalization event is detected, is indicated (mean ± SD; n = 5; 298 cells analyzed). Scale bar, 5 μm. ( B ) Quantification of the number of TERRA-hTR colocalizations per nucleus (mean ± SD; n = 5; 298 cells analyzed). ( C ) Detection of TERRA, hTR, and telomeres by smiFISH/RAP1 IF in HeLa cells (mean ± SD; n = 2; 102 cells analyzed). Scale bar, 5 μm. ( D ) Quantification of the number of TERRA-hTR colocalizations per cell detected at telomeres (TERRA-hTR-RAP1) and outside telomeres (TERRA-hTR w/o RAP1) (mean ± SD; n = 2; 102 cells analyzed). ( E ) Number of hTR-RAP1 colocalizations per cell with and w/o TERRA (mean ± SD; n = 2; 102 cells analyzed). ( F ) Quantification of the number of TERRA-hTR foci at telomeres (TERRA-hTR-RAP1) and outside telomeres (TERRA-hTR w/o RAP1) during G 1 , S, and G 2 phase in HeLa cells upon cell synchronization (mean ± SD; n = 2; total number of cells analyzed: 54 (G 1 phase), 46 (S phase), and 45 (G 2 phase). Fraction of hTR-TERRA foci at telomeres: 20.9 ± 3.3% in G 1 -phase cells, 40.2 ± 11% in S-phase cells, and 41.1 ± 5.5% in G 2 -phase cells.

Article Snippet: Cells were blocked by incubation in 1× PBG buffer (0.2% fish gelatin, 0.5% BSA, 1× PBS) for 1 to 6 hours and incubated for 1 hour with anti-RAP1 primary antibody (rabbit anti–TERF2-IP antibody, from Novus Biological, catalog no. NB100-292; RRID: AB_10000825) diluted 1:500 in 1× PBG.

Techniques:

Journal: Science Advances

Article Title: TERRA transcripts localize at long telomeres to regulate telomerase access to chromosome ends

doi: 10.1126/sciadv.adk4387

Figure Lengend Snippet: ( A ) Telomere length measurement by TRF through Southern blot in HeLa cells in the indicated conditions. NT, untreated; CTR, DMSO-treated. Estimated average telomere length and elongation rate for each sample are indicated in the table below. Average ± SD from two technical replicates. ( B ) Detection of TERRA and telomeres by smiFISH/IF in CTR, 7PD rescue, and 24PD rescue cells. Scale bar, 5 μm. ( C ) Quantification of the number of telomeric TERRA foci detected per nucleus by smiFISH/IF (each dot represents a nucleus) (mean ± SD; n = 2; 124 CTR cells, 111 7PD rescue cells, and 112 24PD rescue cells analyzed). Unpaired nonparametric Kruskal-Wallis test coupled with post hoc Dunn’s multiple-comparison test: ** P < 0.01, *** P < 0.001. ( D and E ) Distribution analyses of the number of telomeric TERRA foci per cell. Two-way analysis of variance (ANOVA) test: ** P < 0.01. Post hoc Tukey’s multiple-comparison test: group 1 to 4 P values = 0.07 (CTR versus 7PD rescue), 0.04 (7PD rescue versus 24PD rescue), not significant (ns) (CTR versus 24PD rescue). ( F ) Detection of TERRA, hTR, and telomeres by smiFISH/IF in CTR, 7PD rescue, and 24PD rescue cells. Scale bar, 5 μm. ( G ) Quantification of the percentage of TERRA-hTR foci colocalizing at telomeres and extratelomeric (mean ± SD; n = 2; 124 CTR cells, 111 7PDs rescue cells, and 112 24PDs rescue cells analyzed). Two-way ANOVA test: **** P < 0.0001. ( H ) Distribution analyses of the number of TERRA-hTR-RAP1 colocalizing foci per nucleus. Two-way ANOVA test: **** P < 0.0001; multiple-comparison test: P = 0.0003 for 7PD versus 24PD rescue, P < 0.0001 for CTR versus 7PD rescue, P = 0.001 CTR versus 24PD rescue. ( I ) Number of telomeric hTR foci not colocalizing with TERRA per cell detected by smiFISH/IF. Kruskal-Wallis test: P = 0.0025.

Article Snippet: Cells were blocked by incubation in 1× PBG buffer (0.2% fish gelatin, 0.5% BSA, 1× PBS) for 1 to 6 hours and incubated for 1 hour with anti-RAP1 primary antibody (rabbit anti–TERF2-IP antibody, from Novus Biological, catalog no. NB100-292; RRID: AB_10000825) diluted 1:500 in 1× PBG.

Techniques: Southern Blot, Comparison

Journal: Science Advances

Article Title: TERRA transcripts localize at long telomeres to regulate telomerase access to chromosome ends

doi: 10.1126/sciadv.adk4387

Figure Lengend Snippet: ( A ) Detection of TERRA and RAP1 by smiFISH/IF in HeLa cells. An example of a colocalization event between TERRA and RAP1 is shown in the image magnifications. DAPI is used to stain nuclei. Scale bar, 5 μm. ( B ) Integrated density quantification of RAP1 foci colocalizing and not colocalizing with TERRA foci in HeLa cells as detected by smiFISH/IF. Each dot represents a single RAP1 focus. Mean ± SD is shown. A total of 110 cells were analyzed in three independent biological replicates. Statistical significance was assessed by Mann-Whitney test. **** P < 0.0001. ( C ) Integrated density quantification of RAP1 foci colocalizing and not colocalizing with TERRA foci in the indicated samples. Each dot represents a single RAP1 focus. Mean ± SD is shown from the following number of samples and biological replicates: 64 CTR cells ( n = 2), 67 BIBR1532 cells (123PDs of treatment with BIBR 1532) ( n = 2), 111 7PD rescue cells ( n = 2), 151 POT1 WT cells ( n = 3), and 148 POT1-ΔOB cells ( n = 3). The Mann-Whitney test was used to assess statistical significance. **** P < 0.0001.

Article Snippet: Cells were blocked by incubation in 1× PBG buffer (0.2% fish gelatin, 0.5% BSA, 1× PBS) for 1 to 6 hours and incubated for 1 hour with anti-RAP1 primary antibody (rabbit anti–TERF2-IP antibody, from Novus Biological, catalog no. NB100-292; RRID: AB_10000825) diluted 1:500 in 1× PBG.

Techniques: Staining, MANN-WHITNEY

Journal: Science Advances

Article Title: TERRA transcripts localize at long telomeres to regulate telomerase access to chromosome ends

doi: 10.1126/sciadv.adk4387

Figure Lengend Snippet: ( A ) Detection of TERRA and telomeres in HeLa cells by smiFISH/IF. Image acquisitions were performed using three-dimensional structured illumination microscopy (3D-SIM). Examples of TERRA foci colocalizing with a single telomere (top images) or telomere doublet (bottom images) are displayed. TERRA is shown in red; telomeres are in green. Scale bar is indicated in the rotated view images. ( B ) Quantification of the fraction of single telomeres versus telomere doublets colocalizing with TERRA. Data are shown as percentage of TERRA-colocalizing telomeres and represent mean ± SD from three independent biological replicates for a total of 30 cells and 663 TERRA-colocalizing RAP1 foci analyzed. ( C and D ) Quantification of the integrated density (C) and volume (D) of RAP1 foci colocalizing (with TERRA) and not colocalizing (without TERRA) with TERRA. Both TERRA-single telomere and TERRA-telomere doublet colocalizations were considered. Data are shown as arbitrary units (a.u.), in (C), and μm 3 , in (D), and represents mean ± SD from three independent biological replicates for a total of 30 cells and 663 TERRA-colocalizing RAP1 foci analyzed. The Mann-Whitney test was used to assess statistical significance. **** P < 0.0001. ( E ) Quantification of the integrated density of all TRF1-mCherry foci and TRF1-mCherry foci colocalizing with MS2-tagged telomere 15q TERRA transcripts per nucleus. Forty nuclei corresponding to 3690 telomeres and 73 telomeres colocalizing with MS2-TERRA transcripts were analyzed from imaging datasets obtained in . Statistical analysis was performed with a Kolmogorov-Smirnov test: P ≤ 0.0001.

Article Snippet: Cells were blocked by incubation in 1× PBG buffer (0.2% fish gelatin, 0.5% BSA, 1× PBS) for 1 to 6 hours and incubated for 1 hour with anti-RAP1 primary antibody (rabbit anti–TERF2-IP antibody, from Novus Biological, catalog no. NB100-292; RRID: AB_10000825) diluted 1:500 in 1× PBG.

Techniques: Microscopy, MANN-WHITNEY, Imaging

Journal: Science Advances

Article Title: TERRA transcripts localize at long telomeres to regulate telomerase access to chromosome ends

doi: 10.1126/sciadv.adk4387

Figure Lengend Snippet: ( A ) Northern blot analysis of TERRA in HeLa cells upon TERRA-ASO or control ASO (ASO SCR) transfection. Bottom image shows 18 S rRNA band upon gel run. ( B ) Quantification of TERRA signal from Northern blot analyses of TERRA-ASO–transfected cells shown as fold over ASO SCR (dashed line). * P < 0.05; mean ± SD, n = 2. ( C ) RT-qPCR analyses of TERRA expression from the indicated telomeres in TERRA-ASO cells shown as fold over ASO SCR. Mean ± SD from four independent biological replicates. Unpaired t test: ** P < 0.01, *** P < 0.001. ( D ) Integrated density quantification of TERRA foci colocalizing with RAP1 foci. Each dot represents a single TERRA signal (mean ± SD; n = 2; 131 ASO SCR cells and 122 TERRA-ASO cells analyzed). Mann-Whitney test: ** P < 0.01. ( E ) Quantification of the number of hTR foci detected per nucleus in TERRA-ASO and ASO SCR cells (mean ± SD; n = 2; 131 ASO SCR cells and 122 TERRA-ASO cells analyzed). Mann-Whitney test: **** P < 0.0001. ( F ) RT-qPCR quantification of hTR levels using primer pairs detecting the precursor or mature RNA ( , ). Results are shown as fold change over ASO SCR (dashed line) (mean ± SD, n = 2). U6 gene was used for normalization . ( G ) Detection of hTR and telomeres by smiFISH/IF. Scale bar, 5 μm. ( H ) Quantification of the number of telomeric hTR foci detected per nucleus. Data are shown as number of RAP1-hTR colocalizations per cell (each dot represents a cell) (mean ± SD, n = 2; 131 ASO SCR cells and 122 TERRA-ASO cells analyzed). Mann-Whitney test: **** P < 0.0001. ( I and J ) Distribution analysis of the number of RAP1-hTR colocalizations detected per cell. Two-way ANOVA test: ** P < 0.01.

Article Snippet: Cells were blocked by incubation in 1× PBG buffer (0.2% fish gelatin, 0.5% BSA, 1× PBS) for 1 to 6 hours and incubated for 1 hour with anti-RAP1 primary antibody (rabbit anti–TERF2-IP antibody, from Novus Biological, catalog no. NB100-292; RRID: AB_10000825) diluted 1:500 in 1× PBG.

Techniques: Northern Blot, Control, Transfection, Quantitative RT-PCR, Expressing, MANN-WHITNEY

Journal: bioRxiv

Article Title: Crk proteins activate the Rap1 guanine nucleotide exchange factor C3G by segregated adaptor-dependent and -independent mechanisms

doi: 10.1101/2022.11.24.515150

Figure Lengend Snippet: ( A ) Schematic representation of the PRM mutants used to study the activation of C3G by CrkL. ( B ) Representative exchange reactions of Rap1:mant-dGDP (200 nM) catalyzed by C3G wild type and mutants (1 µM) in the presence of CrkL (40 µM). Lines are the single exponential decay models fitted to obtain the k obs . ( C ) Nucleotide exchange rates of C3G wild type and mutants (1 µM) alone and in the presence of 40 µM CrkL. Data are shown as scatter plots with bars, means ± standard deviation. The number of independent measurements is indicated in parentheses. Statistical comparison was analyzed using ANOVA followed by Tukey’s multiple comparisons test; *** P < 0.001, **** P < 0.0001, ns P > 0.05. ( D ) Dose-dependent effect of CrkL on the GEF activity of C3G (1 µM) wild type and two mutants. Lines are the fitted sigmoidal models. ( E ) Nucleotide exchange rates of Src-phosphorylated C3G (pC3G, 0.2 µM) wild type and mutants, alone and in the presence of 5 µM CrkL. Statistical comparisons were done as in C. ( F ) Dose-dependent effect of CrkL on the GEF activity of pC3G (0.2 µM) wild type and mutants. Dissociation rate constants in C-F are referred to 1 µM C3G for comparison.

Article Snippet: The following primary antibodies were used for western blot, at 1/1000 dilutions unless otherwise specified: mouse monoclonal antibody (mAb) G9 against C3G (sc-393836), rabbit polyclonal antibody (pAb) C-20 against CrkL (sc-319), mouse mAb B-2 against GFP (sc-9996), rabbit pAb 121 against Rap1 (sc-65) were from Santa Cruz Biotechnology, mouse mAb 4G10 against phospho-Tyr (05-321, Millipore), and mouse mAb against β-actin (AC-15, Sigma-Aldrich, used at 1/2000).

Techniques: Activation Assay, Standard Deviation, Comparison, Activity Assay

Journal: bioRxiv

Article Title: Crk proteins activate the Rap1 guanine nucleotide exchange factor C3G by segregated adaptor-dependent and -independent mechanisms

doi: 10.1101/2022.11.24.515150

Figure Lengend Snippet: ( A ) Analysis by co-immunoprecipitation (coIP) in Jurkat cells of the interaction between stably expressed exogenous C3G-mEGFP variants and endogenous CrkL. Proteins were immunoprecipitated with affinity resin against GFP. CrkL and mEGFP-tagged proteins were detected in cell lysates and in the IP by western blot (WB). ( B ) Analysis of the interaction between C3G-mEGFP and CrkL in HEK293T cells. mEGFP-tagged C3G WT and mutants were transiently expressed and the interaction with endogenous CrkL was analyzed by coIP as in A. ( C ) Imaging of C3G-mEGFP (upper panels), and cortical actin (stained with phalloidin-iFluor 647, middle panels) in Jurkat cells expressing C3G wild type or the indicated mutants. Nuclei were stained with DAPI. Cells were plated on coverslips treated with poly-L-lysine and were unstimulated (-anti-CD3) or stimulated with OKT-3 antibody against CD3. Representative fields are shown, in which the median of the Manders’ overlap coefficient (MOC) for the cells in each field is similar to the value observed for the total of cells analyzed. Scale bars, 20 μm. ( D ) Quantification of the co-localization of C3G-mEGFP with phalloidin-stained actin in confocal microscopy images of Jurkat cells as shown in C. MOC values are shown as scatter plots (from left to right, n = 637, 466, 346, 565, 423, and 459 cells). Middle bars mark the median and whiskers are the 25th and 75th percentiles. Statistical analysis were done with non-parametric Kruskal-Wallis and Dunn’s multiple comparisons tests (**** P < 0.0001, n.s. P > 0.05). ( E ) Fluorescence intensity of C3G-mEGFP and phalloidin staining along the yellow dashed lines in panel C, which run across representative cells. ( F ) Analysis of Rap1 activation in Jurkat cells stably expressing C3G-mEGFP WT and mutants, or isolated mEGFP as control, before and after stimulation with antibody against CD3.

Article Snippet: The following primary antibodies were used for western blot, at 1/1000 dilutions unless otherwise specified: mouse monoclonal antibody (mAb) G9 against C3G (sc-393836), rabbit polyclonal antibody (pAb) C-20 against CrkL (sc-319), mouse mAb B-2 against GFP (sc-9996), rabbit pAb 121 against Rap1 (sc-65) were from Santa Cruz Biotechnology, mouse mAb 4G10 against phospho-Tyr (05-321, Millipore), and mouse mAb against β-actin (AC-15, Sigma-Aldrich, used at 1/2000).

Techniques: Immunoprecipitation, Stable Transfection, Western Blot, Imaging, Staining, Expressing, Confocal Microscopy, Fluorescence, Activation Assay, Isolation, Control

Journal: Oncotarget

Article Title: Heregulin, a new regulator of telomere length in human cells

doi:

Figure Lengend Snippet: A. Retrovirally-induced HRGβ2 regulates TRF2 and RAP1 expression and telomere length in MCF-7 breast cancer cells. Left. Representative immunoblot ( n = 3 ) showing upregulation of TRF2 and RAP1 in MCF-7 cells retrovirally engineered to overexpress HRGβ 2 . PD 0 represents the first sub-passage after selection for retroviral infection. At the indicated PDs, cells were washed with cold PBS and solubilized in lysis buffer containing phosphatase and protease inhibitors. Fifty μg of protein per sample was resolved by SDS-PAGE and subjected to Western blotting for TRF2 and hRap1 as described above (see “Materials and Methods” for details). Middle panel. MCF-7/HRGβ 2 cells do not exhibit significant changes in telomerase activity as assessed by the TRAP assay. Three thousand cell equivalents were used for each reaction and a representative experiment is shown ( n = 3 ). Right panel. Telomere length changes in MCF-7 cells upon retrovirally-induced HRGβ 2 overexpression. The panel shows a representative genomic blotting analysis of telomeric restriction fragments in Hin fI/ Rsa I-digested genomic DNA from retrovirally-infected MCF-7 cells probed with a TTAGGG repeat fragment. B. HRG knockdown reduces the presence of TRF2 and RAP1 on telomeres and promotes telomere lengthening. Top left. Depletion of HRG with siRNA significantly affects TRF2 and RAP1 protein levels. A representative Western blot of MDA-MB-231 cell lysates 72 h after transfection of siRNA to HRG or siRNA control is shown ( n = 3 ). Top right. TRF length analysis in HRG knockdown MDA-MB-231 cells. The panel shows a representative genomic blotting analysis of telomeric restriction fragments in Hin fI/ Rsa I-digested genomic DNA from MDA-MB-231 cells transiently transfected with control siRNA or graded concentrations of HRG siRNA (PD 4) probed with a TTAGGG repeat fragment ( n = 3 ). Bottom. Reduced TRF2 and RAP1 telomeric signals after HRG siRNA treatment. Western blot analysis showing the significant depletion of HRGβ 2 protein with a specific siRNA. The panel shows representative immunofluorescence images of MDA-MB-231 cells 48 h after introduction of HRG siRNA: staining with anti-TRF2 ( red ) and anti-RAP1 ( green ) antibodies is shown ( n = 3 ). DAPI was used to visualize nuclear DNA ( blue ).

Article Snippet: Primary antibodies against TRF2 (IMG-124), TRF1 (IMG-283) and RAP1 (IMG-272) were purchased from Imgenex (San Diego, CA).

Techniques: Expressing, Western Blot, Selection, Retroviral, Infection, Lysis, SDS Page, Activity Assay, TRAP Assay, Over Expression, Knockdown, Transfection, Control, Immunofluorescence, Staining

Journal: Oncotarget

Article Title: Heregulin, a new regulator of telomere length in human cells

doi:

Figure Lengend Snippet: A. Subnuclear localization of HRGβ 2 and its relationship with telomeric loci. A431 cells naturally overexpressing HRGβ 2 were permeabilized twice and sequentially stained with an antibody against HRGβ 2 ( red ) and the fluorescein-conjugated telomere-specific peptide nucleic acid (PNA) probe ( green ). DAPI was used to visualize nuclear DNA ( blue ). Merged images reveal partial co-localization of endogenous HRGβ 2 and telomeres ( yellow ). B. Representative immunofluorescent images of A431 cells co-stained for endogenous HRGβ 2 ( green ) using anti-TRF2 or anti-RAP1 ( red ) antibodies is shown ( n = 3 ). Merged images, including DAPI staining of DNA, reveal a prominent co-localization ( yellow ) of endogenous HRGβ 2 with TRF2 and RAP1.

Article Snippet: Primary antibodies against TRF2 (IMG-124), TRF1 (IMG-283) and RAP1 (IMG-272) were purchased from Imgenex (San Diego, CA).

Techniques: Staining