polyclonal anti tpp1 antibody  (R&D Systems)

Journal: Cell Reports Medicine

Article Title: Recreating pathophysiology of CLN2 disease and demonstrating reversion by TPP1 gene therapy in hiPSC-derived retinal organoids and retina-on-chip

doi: 10.1016/j.xcrm.2025.102244

Figure Lengend Snippet: Characterization of CLN2 ROs (A) Schematic of the hiPSC lines, RO differentiation protocol, and analysis time points (days 84, 200, and 350) and bright-field image of ROs at day 200. (B and C) Uniform manifold approximation and projection (UMAP) of a single-cell RNA-seq dataset from ROs at day 192 ( n = 2 CTRLs, 2 CLN2s) and (C) cell type composition. (D) UMAP of cell type-specific markers ( GNGT1 : rods; ARR3 : cones; TFAP2A : amacrine cells; CA10 : bipolar cells; ONECUT1 : horizontal cells; RLBP1 : Müller glia; KI67 : proliferative progenitors). (E) Recoverin (photoreceptors) immunostaining in CTRL1 and CLN2 ROs. (F) UMAP of TPP1 gene expression as expression levels (left) and expression density (right). (G) Heatmap of TPP1 expression (counts TPP1 /counts cell ∗10,000) and percentage of TPP1 -expressing cells. (H) TPP1 immunostaining and quantification in CTRL (image: CTRL1) and CLN2 ROs at days 84, 200, and 350. Values were normalized on TPP1 expression in CTRLs. n = 5 ROs, one differentiation. (I) Single confocal plane of TPP1 and recoverin in ROs at day 200. Yellow-dashed square: magnified area in the third column. n = 5 ROs from one differentiation. Graphs shows number of TPP1 punctae per 10 μm 3 . (J) Single confocal plane showing colocalization of TPP1 with LAMP1 at day 350. Arrowhead: examples of colocalizing. Values: mean ± SEM. Scale bars: (A) 200 μm, (E, H) 100 μm, (I) 25 μm. Hoechst: (E, H) blue, (J) gray. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: After incubation, the plate was washed and any TPP1 protein captured by the immobilized antibody was detected by a polyclonal anti-TPP1 antibody (R&D Systems no. AF2237) labeled with SULFO-TAG.

Techniques: RNA Sequencing, Immunostaining, Gene Expression, Expressing

Journal: Cell Reports Medicine

Article Title: Recreating pathophysiology of CLN2 disease and demonstrating reversion by TPP1 gene therapy in hiPSC-derived retinal organoids and retina-on-chip

doi: 10.1016/j.xcrm.2025.102244

Figure Lengend Snippet: Autofluorescence, SCMAS, and lipid accumulation in CLN2 ROs (A) LipidSpot and quantification of lipid droplets per 10 μm 3 at day 200 CTRL (image: CTRL1) and CLN2 ROs. Hoechst: blue. n = 5 ROs from one differentiation. (B) SCMAS immunostaining and quantification in CTRL (image: CTRL1) and CLN2 ROs at days 84, 200, and 350. n = 5 ROs from one differentiation. (C) Single confocal plane showing co-localization of SCMAS and green autofluorescence in day 350 CTRL (image: CTRL1) and CLN2 (image: CLN2-1) ROs. SCMAS and autofluorescent co-localization: white. (D) Single confocal plane showing co-localization of SCMAS with recoverin and CRALBP in CTRL (image: CTRL1) and CLN2 ROs at day 200. Yellow dashed square: magnified area in (D′). (D′) Yellow arrowheads: examples of colocalizing signal. (E) Quantification of SCMAS punctae per 10 μm 3 and SCMAS punctae volume in CTRL (CTRL1, CTRL2) and CLN2 ROs at day 200. n = 5 ROs from one differentiation. (F) Co-localization percentage of SCMAS with recoverin and CRALBP in CTRL (CTRL1, CTRL2) and CLN2 ROs at day 200. n = 5 ROs, one differentiation. Values are mean ± SEM. (A, B) Values normalized to CTRL ROs. Scale bars: (A) 10 μm, (B) 100 μm, (C, D) 25 μm. Hoechst: (A, C) blue, (D, D′) gray. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: After incubation, the plate was washed and any TPP1 protein captured by the immobilized antibody was detected by a polyclonal anti-TPP1 antibody (R&D Systems no. AF2237) labeled with SULFO-TAG.

Techniques: Immunostaining

Journal: Cell Reports Medicine

Article Title: Recreating pathophysiology of CLN2 disease and demonstrating reversion by TPP1 gene therapy in hiPSC-derived retinal organoids and retina-on-chip

doi: 10.1016/j.xcrm.2025.102244

Figure Lengend Snippet: AAV9.hCLN2 treatment can decrease and prevent SCMAS accumulation in CLN2 ROs (A–C) SCMAS immunostaining and quantification of ROs treated with AAV9.hCLN2 at days 88, 123, and 260. AAV9.hCLN2 dose 1: 5 × 10 9 , dose 2: 5 × 10 10 , and dose 3: 1.67 × 10 11 gc/RO. Values were normalized on SCMAS expression in CTRL ROs = dashed line. Number of analyzed RO: see G–4I. (D) Single confocal plane of SCMAS immunostaining and quantification in day 123 + 35 ROs treated with AAV9.hCLN2. N = 5 ROs, two experiments. Values are mean ± SEM. Scale bars: (A–C) 100 μm, (D) 25 μm. Hoechst: blue. Tx: treatment.

Article Snippet: After incubation, the plate was washed and any TPP1 protein captured by the immobilized antibody was detected by a polyclonal anti-TPP1 antibody (R&D Systems no. AF2237) labeled with SULFO-TAG.

Techniques: Immunostaining, Expressing

Journal: Cell Reports Medicine

Article Title: Recreating pathophysiology of CLN2 disease and demonstrating reversion by TPP1 gene therapy in hiPSC-derived retinal organoids and retina-on-chip

doi: 10.1016/j.xcrm.2025.102244

Figure Lengend Snippet: scRNA-seq highlights dysregulation of protein translation and mitochondrial function in CLN2 RO cones (A) Differential gene expression (DGE) analysis performed on the cone cluster of the scRNA-seq dataset ( n = 2 CTRL and 2 CLN2 RO samples). Heatmap shows top 25 up- and downregulated genes sorted by a Bonferroni-corrected p value in individual cells of each line. Notable genes are highlighted in red. (B) Network plot (CNET) of a gene set enrichment analysis (GSEA) comparing Gene Ontology (GO) terms (biological processes, cellular components, and metabolic function) of cones. Node color: adjusted p value of enrichment. Node size: number of genes in the core enrichment set. (C) UCell score of selected GO terms of three clusters (ribosomes, mitochondrial membrane, and respiration) enriched in the GSEA analysis. Color: average-scaled U-score. (D) iRegulon analysis of cone DGE (CLN2s vs. CTRLs). y axis: normalized enrichment score (NES) of each depicted transcription factor in DGE cone dataset. TP53 -selected downstream targets are depicted in the light blue box. (E) RICTOR (regulator of the mTOR complex 2) expression in cones. Adjusted p value: Wilcoxon test and Bonferroni correction. (F) Gene expression heatmap of downstream targets of RICTOR (enriched in a CLN2 brain dataset from Sleat et al., meta-analysis performed by Kline et al.). Red-labeled genes were found significantly different in cones of RO in our dataset. (G and H) Single confocal plane showing TOMM20 with (G) PNA lectin (PNAL) and (H) LAMP2 in ROs at day 158. Scale bars, 20 μm. (I and J) Quantification of TOMM20 signal in the PNAL+ area (I) and TOMM20/LAMP2 co-localization (J). Values are mean ± SEM. n = 14–17 ROs from two differentiations, respectively. (K) Putative dysregulation mechanisms in cones of CLN2 ROs. Hoechst: gray. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: After incubation, the plate was washed and any TPP1 protein captured by the immobilized antibody was detected by a polyclonal anti-TPP1 antibody (R&D Systems no. AF2237) labeled with SULFO-TAG.

Techniques: Gene Expression, Membrane, Expressing, Labeling

Journal: Cell Reports Medicine

Article Title: Recreating pathophysiology of CLN2 disease and demonstrating reversion by TPP1 gene therapy in hiPSC-derived retinal organoids and retina-on-chip

doi: 10.1016/j.xcrm.2025.102244

Figure Lengend Snippet: AAV9.hCLN2 delivery to CLN2 ROs restores TPP1 expression (A) Schematic of AAV9.hCLN2 treatment of ROs. (B and C) UMAP of a single-cell RNA-seq dataset derived from ROs at day 192 ( n = 2 CTRLs, 2 CLN2 patient lines, and 2 AAV9.hCLN2-treated CLN2 patient lines) indicating individual cell types and (C) cell type composition. (D) UMAP of TPP1 transgene expression in AAV9.hCLN2-treated ROs as expression levels and expression density. (E) Heatmaps of TPP1 transgene expression levels (counts TPP1 /counts cell ∗10,000) and the percentage of TPP1 -expressing cells (in %). (F) Transduction efficiency of RO cell types. Top: cell types colored in shades of red proportionally to their TPP1 transgene expression. Ganglion cells (GCs, gray) were not found in day 192 ROs. Bottom: proportional area chart. HCs, horizontal cells; MGs, Müller glia; BCs, bipolar cells; ACs, amacrine cells. (G–I) TPP1 immunostaining and quantification of ROs treated with AAV9.hCLN2 at days 88, 123, and 260. AAV9.hCLN2 dose 1: 5 × 10 9 , dose 2: 5 × 10 10 , and dose 3: 1.67 × 10 11 gc/RO. Values were normalized to CTRL ROs (dashed line). Analyzed ROs: CLN2-1 n = 8–11; CLN2-2 n = 3–8; CTRL1 n = 9–14; CTRL2 n = 8–9. (J) Single confocal plane and quantification of TPP1 in day 123 + 35 ROs treated with AAV9.hCLN2. n = 5 ROs, 2 experiments. (K) TPP1 protein concentration in supernatants in day 123 + 35 ROs treated with AAV9.hCLN2, evaluated by electrochemiluminescence (ECL) immunoassay. Analyzed ROs: CLN2-1 n = 21–22, 3 experiments; CLN2-2 n = 16–18, 5 experiments; CTRL1 n = 32 from 5 experiments; CTRL2 n = 25, 3 experiments. Values are mean ± SEM. Scale bars: (G–I) 100 μm, (J) 25 μm. Hoechst: blue. Tx: treatment. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: After incubation, the plate was washed and any TPP1 protein captured by the immobilized antibody was detected by a polyclonal anti-TPP1 antibody (R&D Systems no. AF2237) labeled with SULFO-TAG.

Techniques: Expressing, RNA Sequencing, Derivative Assay, Transduction, Immunostaining, Protein Concentration, Electrochemiluminescence

Journal: Cell Reports Medicine

Article Title: Recreating pathophysiology of CLN2 disease and demonstrating reversion by TPP1 gene therapy in hiPSC-derived retinal organoids and retina-on-chip

doi: 10.1016/j.xcrm.2025.102244

Figure Lengend Snippet: Characterization and AAV9.hCLN2 treatment of CLN2 RPE cells (A) TPP1 and SCMAS immunostaining and quantification of hiPSC-RPE cultured for 4 weeks. n = 3, one differentiation. (B) Schematics of AAV9.hCLN2 treatment of the hiPSC-RPE. (C and D) TPP1 and SCMAS immunostaining and SCMAS quantification of hiPSC-RPE 63 days after treatment with AAV9.hCLN2. AAV9.hCLN2 dose 1: 10 5 gc/cell and dose 2: 10 6 gc/cell. n = 4–5, one differentiation. Values are mean ± SEM. Scale bars: (A) 25 μm, (C, D) 100 μm. Hoechst: blue. Tx: treatment. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: After incubation, the plate was washed and any TPP1 protein captured by the immobilized antibody was detected by a polyclonal anti-TPP1 antibody (R&D Systems no. AF2237) labeled with SULFO-TAG.

Techniques: Immunostaining, Cell Culture

Journal: Cell Reports Medicine

Article Title: Recreating pathophysiology of CLN2 disease and demonstrating reversion by TPP1 gene therapy in hiPSC-derived retinal organoids and retina-on-chip

doi: 10.1016/j.xcrm.2025.102244

Figure Lengend Snippet: Evaluation of AAV9.hCLN2 gene therapy in CLN2 RoC (A) Schematics of AAV9.hCLN2 treatment of the RoC. (B and C) TPP1 and SCMAS immunostaining and quantification of day 123 + 28 ROs treated with AAV9.hCLN2 in the RoC. AAV9.hCLN2 dose 1: 6.5 × 10 9 , dose 2: 6.5 × 10 10 , and dose 3: 2.17 × 10 11 gc/well. TPP1 and SCMAS intensity in CTRL organoids are represented as dashed line. Analyzed ROs: CLN2-1 n = 10–11; CLN2-2 n = 8; CTRL1 n = 16; CTRL2 n = 14. (D) TPP1 immunostaining and quantification of hiPSC-RPE cells in AAV9.hCLN2-treated RoCs. Number of analyzed RoC wells: CLN2-1, CLN2-2 n = 1; CTRLs n = 4. (E and F) Quantification of TPP1 (E) and SCMAS (F) in ROs treated with AAV9.hCLN2 at day 123 + 35 in RO culture (gray line, treatment, doses, and n , see ) or at day 123 + 28 in RoC (red line, treatment, doses, and n , see B and C). Values were normalized on TPP1 or SCMAS expression in CTRL ROs or RoC = dashed line. (G) TPP1 protein in supernatant of ROs treated with AAV9.hCLN2 in RO culture (gray line) or RoC (red line), evaluated by electrochemiluminescence (ECL) immunoassay. Gray and red dashed lines: average concentration of TPP1 in CTRL samples from RO culture and RoC treatment, respectively. Analyzed RO supernatants: see K. Analyzed ROC supernatants: CLN2-1 n = 7–19, 5 RoC; CLN2-2 n = 9–15, 4 RoC; CTRL1 n = 25, 7 RoC; CTRL2 n = 27, 7 RoC. Scale: log10. Values and dots are mean ± SEM. Scale bars: (B, C) 100 μm, (D) 50 μm. Hoechst: blue. Tx: treatment. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: After incubation, the plate was washed and any TPP1 protein captured by the immobilized antibody was detected by a polyclonal anti-TPP1 antibody (R&D Systems no. AF2237) labeled with SULFO-TAG.

Techniques: Immunostaining, Expressing, Electrochemiluminescence, Concentration Assay

Journal: JCI Insight

Article Title: TPP1 mutagenesis screens unravel shelterin interfaces and functions in hematopoiesis

doi: 10.1172/jci.insight.138059

Figure Lengend Snippet: ( A ) Schematic representation of the interactions between shelterin proteins and telomerase at chromosome ends. ( B ) Sequence alignment of the human TPP1 POT1- and TIN2-binding domains with indicated mammalian orthologs. Residues of human TPP1 that were mutated in this screen are shown above the alignment. TPP1 mutants defective in binding POT1 and TIN2 are highlighted in red. Brackets indicate 2 residues simultaneously mutated (double mutant). Asterisks, colons, and periods beneath the sequence lineups represent identical residues, strongly conserved residues, and weakly conserved residues, respectively, as described by the MUSCLE algorithm. Cylinders underneath the sequence alignment indicate α helices. The structure of the POT1 C-terminus bound to the TPP1-PBD (Protein Data Bank [PDB]: 5UN7) is shown above the TPP1 domain diagram with POT1 shown in gray and TPP1 shown in yellow. Structure of the TIN2 TRFH -TPP1 TBM -TRF2 TBM complex (PBD: 5XYF) is shown below the TPP1 domain diagram with TIN2 TRFH represented in gray, TRF2 TBM represented in purple, and TPP1 TBM represented in pink. TPP1 amino acids whose mutation resulted in POT1- and TIN2-binding defects are shown in red in the structures.

Article Snippet: The following antibodies were used for detection with chemiluminescence by ECL plus reagents (Pierce ECL Western Blotting Substrate; Thermo Fisher Scientific): mouse monoclonal anti-FLAG M2-HRP conjugate (MilliporeSigma; A8592; 1:10,000), mouse monoclonal anti–c-Myc (9E10) HRP conjugate (Santa Cruz Biotechnology; sc-40 HRP; 1:10,000), rabbit polyclonal anti-TPP1 antibody (Bethyl Laboratories, A303-069A, 1:500), and anti-rabbit HRP-conjugated secondary antibody (Jackson ImmunoResearch; 111035045).

Techniques: Sequencing, Binding Assay, Mutagenesis

Journal: JCI Insight

Article Title: TPP1 mutagenesis screens unravel shelterin interfaces and functions in hematopoiesis

doi: 10.1172/jci.insight.138059

Figure Lengend Snippet: ( A ) Pulldown of transiently expressed FLAG-TPP1 PBD mutants on anti-FLAG–conjugated beads with Myc-POT1. ( B ) Pulldown of transiently expressed FLAG-TPP1 C-terminus mutants on anti-FLAG–conjugated beads with Myc-TIN2. These data are representative of at least 2 independent transfections and co-IPs ( n = 2).

Article Snippet: The following antibodies were used for detection with chemiluminescence by ECL plus reagents (Pierce ECL Western Blotting Substrate; Thermo Fisher Scientific): mouse monoclonal anti-FLAG M2-HRP conjugate (MilliporeSigma; A8592; 1:10,000), mouse monoclonal anti–c-Myc (9E10) HRP conjugate (Santa Cruz Biotechnology; sc-40 HRP; 1:10,000), rabbit polyclonal anti-TPP1 antibody (Bethyl Laboratories, A303-069A, 1:500), and anti-rabbit HRP-conjugated secondary antibody (Jackson ImmunoResearch; 111035045).

Techniques: Transfection

Journal: JCI Insight

Article Title: TPP1 mutagenesis screens unravel shelterin interfaces and functions in hematopoiesis

doi: 10.1172/jci.insight.138059

Figure Lengend Snippet: ( A ) Anti-Myc antibody–bound Myc-TIN2 was pulled down on protein A/G agarose beads with transiently expressed FLAG-TRF2 and indicated FLAG-TPP1 construct. ( B ) Sequence conservation of a portion of the TIN2 TRFH domain. TIN2 residues examined in this study are labeled above the sequence alignment, with red denoting amino acids whose mutation impaired the TPP1-TIN2 interaction and black denoting amino acids whose mutation did not affect TPP1-TIN2 binding. Asterisks, colons, and periods beneath the sequence lineups represent identical residues, strongly conserved residues, and weakly conserved residues, respectively, as described by the MUSCLE algorithm. ( C ) Pulldown of indicated FLAG-TIN2 construct on anti-FLAG–conjugated beads with WT Myc-TRF2 and Myc-TPP1. ( D ) Structure of the TIN2 TRFH (gray)-TPP1 TBM (pink)- TRF2 TBM (lilac) complex (PDB: 5XYF, ref. ). TIN2 residues whose mutation impacts binding to TPP1 and that likely contact the TPP1 TBM-ext are shown in yellow, TIN2 K160A is shown in green, and TPP1 TBM mutants are shown in red. Data in A and C are representative of at least 5 independent transfections and coimmunoprecipitations (co-IPs) ( n = 5).

Article Snippet: The following antibodies were used for detection with chemiluminescence by ECL plus reagents (Pierce ECL Western Blotting Substrate; Thermo Fisher Scientific): mouse monoclonal anti-FLAG M2-HRP conjugate (MilliporeSigma; A8592; 1:10,000), mouse monoclonal anti–c-Myc (9E10) HRP conjugate (Santa Cruz Biotechnology; sc-40 HRP; 1:10,000), rabbit polyclonal anti-TPP1 antibody (Bethyl Laboratories, A303-069A, 1:500), and anti-rabbit HRP-conjugated secondary antibody (Jackson ImmunoResearch; 111035045).

Techniques: Construct, Sequencing, Labeling, Mutagenesis, Binding Assay, Transfection

Journal: JCI Insight

Article Title: TPP1 mutagenesis screens unravel shelterin interfaces and functions in hematopoiesis

doi: 10.1172/jci.insight.138059

Figure Lengend Snippet: ( A ) Schematic of representative TPP1 PBD (L191A/L193A), TPP1 TBM (Y376A/Y378A), or TPP1 TBM-ext (R393A/L394A) mutations in mice, with their equivalent in humans. ( B ) Experimental scheme. BM was harvested from 5-fluorouracil–treated (5-FU–treated) Mx-Cre + Acd fl/– B6-CD45.2 mice and retrovirally cotransduced with NUP98-HOXA10HD-mCherry and a TPP1 rescue construct expressing an EGFP reporter (vs. EGFP only). Transduced BM progenitors were transplanted into lethally irradiated congenic B6-CD45.1 recipients. Six weeks later, endogenous Acd was inactivated via poly(I:C) administration to induce Mx-Cre expression. ( C ) Survival and ( D ) complete blood counts of transplanted mice at days 6, 13, and 34. n = 10 per group; remaining numbers of mice are noted in D . *** P < 0.001 by log-rank Mantel-Cox test ( C ). ** P < 0.01, *** P < 0.001 by 2-way ANOVA with post hoc Tukey’s test to assess differences in means in D . Mean and 1 SD reported.

Article Snippet: The following antibodies were used for detection with chemiluminescence by ECL plus reagents (Pierce ECL Western Blotting Substrate; Thermo Fisher Scientific): mouse monoclonal anti-FLAG M2-HRP conjugate (MilliporeSigma; A8592; 1:10,000), mouse monoclonal anti–c-Myc (9E10) HRP conjugate (Santa Cruz Biotechnology; sc-40 HRP; 1:10,000), rabbit polyclonal anti-TPP1 antibody (Bethyl Laboratories, A303-069A, 1:500), and anti-rabbit HRP-conjugated secondary antibody (Jackson ImmunoResearch; 111035045).

Techniques: Construct, Expressing, Irradiation

Journal: JCI Insight

Article Title: TPP1 mutagenesis screens unravel shelterin interfaces and functions in hematopoiesis

doi: 10.1172/jci.insight.138059

Figure Lengend Snippet: ( A ) Donor-derived NUP98-HOXA10HD-mCherry + CD11b + Gr-1 + myeloid cells were assessed by flow cytometric analysis of EGFP before poly(I:C)-induced loss of endogenous Acd (d0) or at day 6 after poly(I:C) induction. EGFP reports expression of WT TPP1 versus TPP1 mutants versus EGFP only control (MigR1). ( B ) Relative enrichment of EGFP + myeloid cells at day 6 and 13 is noted and summarized. *** P < 0.001 by 2-way ANOVA with post hoc Tukey’s test to assess differences in means ( B ). n = 10 per group.

Article Snippet: The following antibodies were used for detection with chemiluminescence by ECL plus reagents (Pierce ECL Western Blotting Substrate; Thermo Fisher Scientific): mouse monoclonal anti-FLAG M2-HRP conjugate (MilliporeSigma; A8592; 1:10,000), mouse monoclonal anti–c-Myc (9E10) HRP conjugate (Santa Cruz Biotechnology; sc-40 HRP; 1:10,000), rabbit polyclonal anti-TPP1 antibody (Bethyl Laboratories, A303-069A, 1:500), and anti-rabbit HRP-conjugated secondary antibody (Jackson ImmunoResearch; 111035045).

Techniques: Derivative Assay, Expressing, Control

Journal: JCI Insight

Article Title: TPP1 mutagenesis screens unravel shelterin interfaces and functions in hematopoiesis

doi: 10.1172/jci.insight.138059

Figure Lengend Snippet: ( A ) Immunoblot analysis of the indicated Acd fl/fl MEF cell lines showing reduction of TPP1 protein levels 72 hours after Cre-ER activation with 4-hydroxytamoxifen (4-OHT) and a uniform rescue of protein levels upon infection with indicated TPP1 WT or mutant lentiviruses. Asterisk indicates nonspecific bands detected by the antibody that serve as loading controls. n = 1. ( B ) TIF analysis was performed on the cell lines described in A using peptide nucleic acid–FISH for telomeres (red) and immunofluorescence for 53BP1 (green). DAPI was used to stain the nucleus (blue). Appearance of orange foci in the “Merge” panel indicates TIFs. Arrowheads point to 2 representative TIFs in the panel. ( C ) Quantitation of TIF data of which B is representative. Mean and SD for n = 3 sets of images (each set containing 15–20 cells) are plotted for the indicated cell lines. * P ≤ 0.05 using 2-tailed Student’s t test.

Article Snippet: The following antibodies were used for detection with chemiluminescence by ECL plus reagents (Pierce ECL Western Blotting Substrate; Thermo Fisher Scientific): mouse monoclonal anti-FLAG M2-HRP conjugate (MilliporeSigma; A8592; 1:10,000), mouse monoclonal anti–c-Myc (9E10) HRP conjugate (Santa Cruz Biotechnology; sc-40 HRP; 1:10,000), rabbit polyclonal anti-TPP1 antibody (Bethyl Laboratories, A303-069A, 1:500), and anti-rabbit HRP-conjugated secondary antibody (Jackson ImmunoResearch; 111035045).

Techniques: Western Blot, Activation Assay, Infection, Mutagenesis, Immunofluorescence, Staining, Quantitation Assay

Journal: JCI Insight

Article Title: TPP1 mutagenesis screens unravel shelterin interfaces and functions in hematopoiesis

doi: 10.1172/jci.insight.138059

Figure Lengend Snippet: ( A ) Schematic of human mutations and their equivalents in mice. ( B ) Experimental scheme, similar to . ( C ) Survival of mice reconstituted with BM containing TPP1 rescue constructs noted in B . ( D ) EGFP expression in donor NUP98-HOXA10HD-mCherry + CD11b + Gr-1 + myeloid cells before poly(I:C)-induced loss of endogenous TPP1 (d0) and subsequent time points. ( E and F ) EGFP expression in donor NUP98-HOXA10HD-mCherry + LSK progenitor cells from BM at day 239 after poly(I:C) induction. n = 10 per group. *** P < 0.001 by log-rank Mantel-Cox test ( C ). * P < 0.05, *** P < 0.001 by 2-way ANOVA with post hoc Tukey’s test to assess differences in means ( D ). Mean and 1 SD reported.

Article Snippet: The following antibodies were used for detection with chemiluminescence by ECL plus reagents (Pierce ECL Western Blotting Substrate; Thermo Fisher Scientific): mouse monoclonal anti-FLAG M2-HRP conjugate (MilliporeSigma; A8592; 1:10,000), mouse monoclonal anti–c-Myc (9E10) HRP conjugate (Santa Cruz Biotechnology; sc-40 HRP; 1:10,000), rabbit polyclonal anti-TPP1 antibody (Bethyl Laboratories, A303-069A, 1:500), and anti-rabbit HRP-conjugated secondary antibody (Jackson ImmunoResearch; 111035045).

Techniques: Construct, Expressing

Journal: Cell reports

Article Title: Two Separation-of-Function Isoforms of Human TPP1 Dictate Telomerase Regulation in Somatic and Germ Cells

doi: 10.1016/j.celrep.2019.05.073

Figure Lengend Snippet: (A) Schematic showing a TPP1-centric view of the shelterin complex in humans.

Article Snippet: The conspicuous abundance of TPP1-L-specific reads in testes relative to other tissues is highlighted with a gray oval. (B) Schematic for the different stages of spermatogenesis. (C) Archived RNA-seq data reveal an increase in TPP1-L abundance in human round spermatids relative to spermatocytes. (D) Immunoblot analysis using an anti-rabbit TPP1 polyclonal antibody (Bethyl Labs) of the chromatin-associated fraction of cells from a biopsy of a human testis reveals a band corresponding to TPP1-L. FLAG-tagged TPP1-L and TPP1-S overexpression cell extracts were run in parallel as size markers. (E) RNAscope staining for TERT mRNA in formaldehyde fixed-paraffin embedded (FFPE) sections of human testes tissue.

Techniques:

Journal: Cell reports

Article Title: Two Separation-of-Function Isoforms of Human TPP1 Dictate Telomerase Regulation in Somatic and Germ Cells

doi: 10.1016/j.celrep.2019.05.073

Figure Lengend Snippet: (A) HeLa-EM2-11ht cells expressing TPP1-S, TPP1-L M87A, or TPP1ΔNOB were analyzed for telomerase recruitment to telomeres by immunofluorescence (IF) and fluorescence in situ hybridization (FISH). “FLAG (TPP1)” indicates IF signal of the indicated TPP1 construct at telomeres (green). Telomerase RNA (“TR”) was detected by FISH with a fluorescently tagged DNA probe (red). The “merge” panel depicts the extent of telomerase recruitment to telomeres (yellow).

Article Snippet: The conspicuous abundance of TPP1-L-specific reads in testes relative to other tissues is highlighted with a gray oval. (B) Schematic for the different stages of spermatogenesis. (C) Archived RNA-seq data reveal an increase in TPP1-L abundance in human round spermatids relative to spermatocytes. (D) Immunoblot analysis using an anti-rabbit TPP1 polyclonal antibody (Bethyl Labs) of the chromatin-associated fraction of cells from a biopsy of a human testis reveals a band corresponding to TPP1-L. FLAG-tagged TPP1-L and TPP1-S overexpression cell extracts were run in parallel as size markers. (E) RNAscope staining for TERT mRNA in formaldehyde fixed-paraffin embedded (FFPE) sections of human testes tissue.

Techniques: Expressing, Immunofluorescence, Fluorescence, In Situ Hybridization, Construct

Journal: Cell reports

Article Title: Two Separation-of-Function Isoforms of Human TPP1 Dictate Telomerase Regulation in Somatic and Germ Cells

doi: 10.1016/j.celrep.2019.05.073

Figure Lengend Snippet: (A) Cartoon representation of contrasting outcomes of the in vivo IF-coFISH telomere extension assay involving FLAG-TPP1 (green), TR (cyan), and newly synthesized mutant telomeric repeats “mutant telomere” (red). White foci in merge of TR and mutant telomere signals depict colocalization.

Article Snippet: The conspicuous abundance of TPP1-L-specific reads in testes relative to other tissues is highlighted with a gray oval. (B) Schematic for the different stages of spermatogenesis. (C) Archived RNA-seq data reveal an increase in TPP1-L abundance in human round spermatids relative to spermatocytes. (D) Immunoblot analysis using an anti-rabbit TPP1 polyclonal antibody (Bethyl Labs) of the chromatin-associated fraction of cells from a biopsy of a human testis reveals a band corresponding to TPP1-L. FLAG-tagged TPP1-L and TPP1-S overexpression cell extracts were run in parallel as size markers. (E) RNAscope staining for TERT mRNA in formaldehyde fixed-paraffin embedded (FFPE) sections of human testes tissue.

Techniques: In Vivo, Synthesized, Mutagenesis

Journal: Cell reports

Article Title: Two Separation-of-Function Isoforms of Human TPP1 Dictate Telomerase Regulation in Somatic and Germ Cells

doi: 10.1016/j.celrep.2019.05.073

Figure Lengend Snippet: (A) Existing high-throughput sequencing information for the ACD locus was visualized using the UCSC genome browser. Vertical gray bar denotes the 5′ end of TPP1-S mRNA. See also Figure S4A and STAR Methods.

Article Snippet: The conspicuous abundance of TPP1-L-specific reads in testes relative to other tissues is highlighted with a gray oval. (B) Schematic for the different stages of spermatogenesis. (C) Archived RNA-seq data reveal an increase in TPP1-L abundance in human round spermatids relative to spermatocytes. (D) Immunoblot analysis using an anti-rabbit TPP1 polyclonal antibody (Bethyl Labs) of the chromatin-associated fraction of cells from a biopsy of a human testis reveals a band corresponding to TPP1-L. FLAG-tagged TPP1-L and TPP1-S overexpression cell extracts were run in parallel as size markers. (E) RNAscope staining for TERT mRNA in formaldehyde fixed-paraffin embedded (FFPE) sections of human testes tissue.

Techniques: Next-Generation Sequencing

Journal: Cell reports

Article Title: Two Separation-of-Function Isoforms of Human TPP1 Dictate Telomerase Regulation in Somatic and Germ Cells

doi: 10.1016/j.celrep.2019.05.073

Figure Lengend Snippet: (A) Deposited Burge lab RNA-seq data from the indicated tissues or organs were visualized on the UCSC genome browser. The conspicuous abundance of TPP1-L-specific reads in testes relative to other tissues is highlighted with a gray oval.

Article Snippet: The conspicuous abundance of TPP1-L-specific reads in testes relative to other tissues is highlighted with a gray oval. (B) Schematic for the different stages of spermatogenesis. (C) Archived RNA-seq data reveal an increase in TPP1-L abundance in human round spermatids relative to spermatocytes. (D) Immunoblot analysis using an anti-rabbit TPP1 polyclonal antibody (Bethyl Labs) of the chromatin-associated fraction of cells from a biopsy of a human testis reveals a band corresponding to TPP1-L. FLAG-tagged TPP1-L and TPP1-S overexpression cell extracts were run in parallel as size markers. (E) RNAscope staining for TERT mRNA in formaldehyde fixed-paraffin embedded (FFPE) sections of human testes tissue.

Techniques: RNA Sequencing

Journal: Cell reports

Article Title: Two Separation-of-Function Isoforms of Human TPP1 Dictate Telomerase Regulation in Somatic and Germ Cells

doi: 10.1016/j.celrep.2019.05.073

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The conspicuous abundance of TPP1-L-specific reads in testes relative to other tissues is highlighted with a gray oval. (B) Schematic for the different stages of spermatogenesis. (C) Archived RNA-seq data reveal an increase in TPP1-L abundance in human round spermatids relative to spermatocytes. (D) Immunoblot analysis using an anti-rabbit TPP1 polyclonal antibody (Bethyl Labs) of the chromatin-associated fraction of cells from a biopsy of a human testis reveals a band corresponding to TPP1-L. FLAG-tagged TPP1-L and TPP1-S overexpression cell extracts were run in parallel as size markers. (E) RNAscope staining for TERT mRNA in formaldehyde fixed-paraffin embedded (FFPE) sections of human testes tissue.

Techniques: Virus, Recombinant, Genome Wide, RNA Sequencing, CRISPR, FLAG-tag, Primer Extension Assay, Software, Mutagenesis, RNAscope, Negative Control, Positive Control