Journal: Aging cell

Article Title: Akt Regulates TPP1 Homodimerization and Telomere Protection

doi: 10.1111/acel.12137

Figure Lengend Snippet: (A) HTC75 cells treated with triciribine or mock treated with DMSO for 3 hours were analyzed by western blotting using the indicated antibodies. Actin was used as loading control. Arrows indicate the two POT1 isoforms. (B) Cells from (A) were examined by telomere chromatin immunoprecipitation (ChIP) using antibodies against TPP1, POT1 and RAP1, followed by dot-blotting with probes against telomere sequences or Alu repeats. Rabbit IgG served as control. (C) Quantification of data from (B) (two independent experiments). ChIP signal intensities were normalized against input DNA. Error bars represent s.d. P-values were determined by the Student t-test.

Article Snippet: The antibodies used for ChIP are: rabbit polyclonal POT1 (N-terminal) ( O'Connor et al., 2006 ), rabbit polyclonal TPP1 (467) ( Liu et al., 2004 ), polyclonal anti-RAP1 (Bethyl Laboratories) and rabbit IgG (Santa Cruz, SC2027).

Techniques: Western Blot, Control, Chromatin Immunoprecipitation

Journal: Aging cell

Article Title: Akt Regulates TPP1 Homodimerization and Telomere Protection

doi: 10.1111/acel.12137

Figure Lengend Snippet: (A) HTC75 cells co-expressing YFPn-tagged TPP1 with YFPc-tagged TPP1 or TIN2 were examined by BiFC assays. The percentage of cells displaying fluorescence complementation was quantitated by flow cytometry. Error bars indicate standard error (n=3). (B) 293T cells transiently co-expressing Myc-tagged TPP1 with Flag-tagged TPP1 or TIN2 were immunoprecipitated with anti-Flag antibodies. The immunoprecipitates were western blotted as indicated. (C) 293T cells transiently co-expressing Myc-tagged TPP1 with Flag-tagged TPP1 or TPP1 OB-fold deletion mutant (TPP1ΔOB) were immunoprecipitated with anti-Flag antibodies. The immunoprecipitates were western blotted as indicated. (D) Bacterially purified GST-tagged TPP1 OB fold only mutant (TPP1 OB) was incubated with MBP-tagged TPP1 OB for GST pull-down assays. The precipitates were resolved by SDS-PAGE and visualized by Coomassie staining or western blotting. GST-tagged Raf-1 Ras-binding domain (RBD) and MBP-tagged FAM118B were used as controls. (E) HTC75 cells were transfected with siRNA oligos against Akt1 (siAKT1-1) in combination with Flag-tagged wildtype TPP1 or TPP1 OB fold deletion mutant (TPP1ΔOB), and then analyzed by Western blotting using the indicated antibodies. Actin was used as loading control. (F) Cells from (E) were examined by immunostaining using anti-53BP1 (red) and TRF2 (green) antibodies. Arrows indicate overlapping foci. (G) Quantification of data from (F). Only cells with >4 co-localized foci were scored. Error bars indicate s.e.m. (n=3). P-values were determined by the Student t-test.

Article Snippet: The antibodies used for ChIP are: rabbit polyclonal POT1 (N-terminal) ( O'Connor et al., 2006 ), rabbit polyclonal TPP1 (467) ( Liu et al., 2004 ), polyclonal anti-RAP1 (Bethyl Laboratories) and rabbit IgG (Santa Cruz, SC2027).

Techniques: Expressing, Fluorescence, Flow Cytometry, Immunoprecipitation, Western Blot, Mutagenesis, Purification, Incubation, SDS Page, Staining, Binding Assay, Transfection, Control, Immunostaining

Journal: Aging cell

Article Title: Akt Regulates TPP1 Homodimerization and Telomere Protection

doi: 10.1111/acel.12137

Figure Lengend Snippet: (A) HTC75 cells co-expressing YFPn-tagged TPP1 with YFPc-tagged TPP1 or TIN2 were either serum starved (0.01% FBS) or treated with triciribine, and then examined by BiFC assays. Error bars indicate standard error (n=3). (B) 293T cells transiently co-expressing TPP1-Flag with Myc-TPP1 were treated with DMSO or triciribine. Cells co-expressing SFB-Akt1 and Myc-TPP1 were also examined. Cell lysates were immunoprecipitated with anti-Flag antibodies and blotted as indicated. (C) HTC75 cells co-expressing YFPn-TPP1 with YFPc-TPP1 were transiently transfected with siRNA oligos against Akt1 and analyzed in BiFC assays. Error bars indicate standard errors (n=3). (D) Cells from (C) were examined by western blotting for Akt1 knockdown efficiency. Actin was used as loading control. (E) HTC75 cells co-expressing YFPn-TPP1 and YFPc-TPP1 were transiently co-transfected with control oligos or siRNA oligos against Akt1 in combination with siRNA-resistant SFB-tagged wildtype (WT) Akt1 or kinase-dead Akt1 (Akt1 K179M). The cells were then examined by western blotting using the indicated antibodies. Actin was used as a loading control. (F) Cells from (E) were analyzed in BiFC assays. P-values were determined by the Student t-test.

Article Snippet: The antibodies used for ChIP are: rabbit polyclonal POT1 (N-terminal) ( O'Connor et al., 2006 ), rabbit polyclonal TPP1 (467) ( Liu et al., 2004 ), polyclonal anti-RAP1 (Bethyl Laboratories) and rabbit IgG (Santa Cruz, SC2027).

Techniques: Expressing, Immunoprecipitation, Transfection, Western Blot, Knockdown, Control

Journal: Oncology reports

Article Title: The transcription factor RFX5 is a transcriptional activator of the TPP1 gene in hepatocellular carcinoma.

doi: 10.3892/or.2016.5240

Figure Lengend Snippet: Figure 2. RFX5 binds to the TPP1 promoter region and activates transcription of the TPP1 promoter. (A) The sketch of the TPP1 promoter region shows the RFX5 ChIP-seq binding peak in HepG2 cell and designed primers. Upper, RFX5 motif consensus sequence from ENCODE is shown, the S-Y motifs from HLA-DRA, HLA-DMA, HLA-DMB and TPP1 are boxed. Lower, (blue bold lines) the design of two PCR amplicons for the luciferase reporter gene, (Box) annotation of the TPP1 promoter based on the ENCODE Histone Modification Tracks, (light blue bold lines) the two ChIP-PCR amplicons and (purple peak) the RFX5 ChIP-seq binding peak in the TPP1 promoter. (B) ChIP-PCR assays showing RFX5 binding to the TPP1 promoter in HCC cells. Immunoprecipitated DNA fragments were analyzed by PCR using two independent primers mapped to the RFX5 ChIP-seq binding peak. (C) The transcriptional activity of two TPP1 promoter reporter constructs. Relative luciferase activity (RLA) was calculated as the ratio of firefly to Renilla luciferase activities to represent the promoter activity and performed in triplicate. The results represent one of three experiments. (D) Western blotting showing RFX5 protein with or without overexpression in 293T cells. (E) Luciferase assay to analyze the transcriptional impact of RFX5 on the TPP1 promoter in HCC cells. *P<0.05, ****P<0.0001.

Article Snippet: Paraffin-embedded tissue slides were heatwd with EDTA (pH 9.0) (ZSGB-BIO, Beijing, China) for antigen retrieval and incubated with rabbit anti-human TPP1 antibody (polyclonal, #12479-1-AP, Proteintech), followed by EnVision detection system (Dako, Denmark).

Techniques: ChIP-sequencing, Binding Assay, Sequencing, Luciferase, Modification, Immunoprecipitation, Activity Assay, Construct, Western Blot, Over Expression

Journal: Oncology reports

Article Title: The transcription factor RFX5 is a transcriptional activator of the TPP1 gene in hepatocellular carcinoma.

doi: 10.3892/or.2016.5240

Figure Lengend Snippet: Figure 3. TPP1 expression is linked to RFX5 expression and poor prognoses in HCC. (A) Real-time PCR showing RFX5 and TPP1 expression in HepG2 cells after RFX5 knockdown. (B) Real-time PCR shows RFX5 and TPP1 expression in HepG2 cells after RFX5 overexpression. (C) TPP1 mRNA expression in the TCGA LIHC data determined by RNAseq. (D) Immunohistochemistry staining of TPP1 in HCC and matched adjacent liver tissues by IHC (magnification, x200). (E) Correlation analysis between RFX5 and TPP1 expression from the TCGA LIHC data. (F) Correlation analysis between RFX5 and TPP1 expression using real-time PCR data from the Guilin cohort. (G) Survival analysis from the overall survival time (OST) in HCC patients with relatively lower or higher TPP1 expression in the tumor tissues. The Kaplan-Meier plot was analyzed by the log-rank test. **P<0.01, ***P<0.001, ****P<0.0001.

Article Snippet: Paraffin-embedded tissue slides were heatwd with EDTA (pH 9.0) (ZSGB-BIO, Beijing, China) for antigen retrieval and incubated with rabbit anti-human TPP1 antibody (polyclonal, #12479-1-AP, Proteintech), followed by EnVision detection system (Dako, Denmark).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Knockdown, Over Expression, Immunohistochemistry, Staining

Journal: Scientific Reports

Article Title: The Fragment HMGA2-sh-3p20 from HMGA2 mRNA 3′UTR Promotes the Growth of Hepatoma Cells by Upregulating HMGA2

doi: 10.1038/s41598-017-02311-0

Figure Lengend Snippet: HMGA2-sh-3p20 upregulates HMGA2 by blocking the TTP-mediated degradation of HMGA2 mRNA. ( A ) The relative expression of HMGA2-sh-3p20 was assessed by qRT-PCR in 35 pairs of clinical HCC tissues and corresponding peritumor tissues ( ***P < 0.001; Wilcoxon’s signed-rank test). ( B ) The correlation between HMGA2 mRNA levels and HMGA2-sh-3p20 levels was measured by qRT-PCR in 30 cases of clinical HCC tissues ( **P < 0.01, r = 0.586; Pearson’s correlation coefficient). ( C ) The luciferase activities of pGL3-HMGA2 were examined by luciferase reporter gene assays in HepG2 cells. ( D ) The expression of HMGA2 was assessed by qRT-PCR and Western blot analysis in Huh7 cells. ( E ) The diagram of TTP-mediated mRNA degradation. ( F ) The diagram of HMGA2-sh-3p20 antagonizes the interaction of TTP with non-hairpin within 3′UTR of HMGA2 mRNA. ( G ) TTP RIP-PCR of HMGA2 in HepG2 cells. ( H ) Effect of TTP on the expression of HMGA2 was measured by qRT-PCR and Western blot analysis in HepG2 cells. ( I ) Effect of HMGA2-sh-3p20 on the expression of TTP-mediated HMGA2 was assessed by qRT-PCR and Western blot analysis in HepG2 cells. The full length blots images are given as Supplementary Fig. . ( J ) TTP RIP-qPCR of HMGA2 in HepG2 cells transfected with HMGA2-sh-3p20. ( K ) Effect of HMGA2-sh-3p20 on the levels of HMGA2 mRNA in HepG2 cells transfected with si-TTP by qRT-PCR. ( L , M ) Effect of HMGA2-sh-3p20 on the half-life of HMGA2 mRNA in HepG2 cells ( L ) or HepG2 cells transfected with si-TTP ( M ) by qRT-PCR. Every experiment was repeated three times. Error bars represent s.d. (n = 3), **p < 0.01; ***p < 0.001 and not significant (NS), Student’s t test.

Article Snippet: The dilution of primary antibody is following: HMGA2 (1: 5000, Genetex), PTEN (1:800, Proteintech), TTP (1:1000, Proteintech), Drosha (1:1000, Proteintech), DGCR8 (1:800, Proteintech), Dicer (1:800, Proteintech), β-actin (1:2000, Abcam).

Techniques: Blocking Assay, Expressing, Quantitative RT-PCR, Luciferase, Western Blot, Transfection

Journal: Scientific Reports

Article Title: The Fragment HMGA2-sh-3p20 from HMGA2 mRNA 3′UTR Promotes the Growth of Hepatoma Cells by Upregulating HMGA2

doi: 10.1038/s41598-017-02311-0

Figure Lengend Snippet: A model shows that the fragment HMGA2-sh-3p20 from HMGA2 mRNA 3′UTR promotes the growth of hepatoma cells by upregulating HMGA2. Bioinformatics analysis shows that 3′UTR of HMGA2 mRNA contains the hairpin structure (HMGA2-sh). Drosha and DGCR8 cleave the HMGA2-sh from the 3′UTR of HMGA2 mRNA, and Dicer contributes to the generation of the HMGA2-sh-3p20 from the HMGA2-sh. Furthermore, HMGA2-sh-3p20 is able to increase the levels of HMGA2 by antagonizing TTP-mediated HMGA2 degradation, while it decreases PTEN by targeting 3′UTR of PTEN mRNA. In addition, the downregulated-PTEN is not able to depress the expression of HMGA2, leading to the upregulation of HMGA2. Functionally, HMGA2-sh-3p20-enhanced HMGA2 accelerates the growth of liver cancer cells.

Article Snippet: The dilution of primary antibody is following: HMGA2 (1: 5000, Genetex), PTEN (1:800, Proteintech), TTP (1:1000, Proteintech), Drosha (1:1000, Proteintech), DGCR8 (1:800, Proteintech), Dicer (1:800, Proteintech), β-actin (1:2000, Abcam).

Techniques: 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: 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: Eye and Brain

Article Title: Peripapillary Retinal Nerve Fiber Layer (pRNFL) Thickness – A Novel Biomarker of Neurodegeneration in Late-Infantile CLN2 Disease

doi: 10.2147/EB.S473408

Figure Lengend Snippet: Heidelberg SD-OCT RNFL analysis of a 9.5-year-old patient with late-infantile CLN2 disease. PRNFL is significantly decreased in the global, temporal superior and temporal inferior pRNFL sectors.

Article Snippet: Currently, intracerebroventricular (ICV) enzyme replacement therapy (ERT) with recombinant human TPP1 (rhTPP1) (cerliponase alfa, Brineura, BioMarin Pharmaceuticals, California, USA) is the only United States Food and Drug Administration (US FDA) and European Medicines Agency (EMA) approved treatment for CLN2 that has proven to decelerate the progression of motor and language function decline.

Techniques: