anti tpp1  (Bethyl)

Journal: Oncology Research

Article Title: Single-Cell and Multi-Omics-Based Characterization of Gastric Cancer Identifies TPP1 as a Potential Target for Gastric Cancer Progression and Treatment

doi: 10.32604/or.2026.070208

Figure Lengend Snippet: This schematic diagram provides an overview of the study’s integrated analytical framework. The workflow begins with data acquisition from public databases, followed by the integration of single-cell RNA sequencing and bulk multi-omics datasets. Metastasis-associated fibroblast subpopulations are then identified through clustering and functional annotation. Key gene modules are extracted using hdWGCNA, and a prognostic risk model is constructed and validated across independent cohorts. Finally, core genes—such as TPP1 —are experimentally validated using in vitro assays to confirm their functional relevance in gastric cancer progression.

Article Snippet: After blocking at room temperature for 1 h, membranes were incubated overnight at 4°C with primary antibodies against TPP1 (1:3000, Proteintech, 25849-1-AP, Chicago, IL, USA) and GAPDH (1:3000, Proteintech, 10494-1-AP, Chicago, IL, USA).

Techniques: Single Cell, RNA Sequencing, Biomarker Discovery, Functional Assay, Construct, In Vitro

Journal: Oncology Research

Article Title: Single-Cell and Multi-Omics-Based Characterization of Gastric Cancer Identifies TPP1 as a Potential Target for Gastric Cancer Progression and Treatment

doi: 10.32604/or.2026.070208

Figure Lengend Snippet: Abnormal expression of TPP1 was verified. ( A ) Boruta algorithm identified six key prognostic signature genes, with TPP1 ranking as the top contributor. ( B ) Pan-cancer differential expression analysis revealed significant overexpression of TPP1 across multiple malignancies. ( C , D ) Bar plots show that TPP1 expression is significantly elevated in gastric cancer tissues based on both unpaired and paired comparisons. ( E ) Kaplan–Meier survival analysis demonstrated that high TPP1 expression correlates with poorer overall survival in gastric cancer patients. ( F ) Immunohistochemical staining confirmed upregulated TPP1 protein expression in gastric cancer tissues (HPA: The Human Protein Atlas). ( G ) Western blot analysis further validated increased TPP1 protein levels in tumor samples. ( H ) qRT-PCR analysis confirmed that TPP1 mRNA expression was significantly elevated in gastric cancer tissues (n = 4). Data are presented as mean ± standard deviation (SD). Statistical comparisons were performed using Student’s t -test unless otherwise indicated. Significance levels were defined as follows: p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****).

Article Snippet: After blocking at room temperature for 1 h, membranes were incubated overnight at 4°C with primary antibodies against TPP1 (1:3000, Proteintech, 25849-1-AP, Chicago, IL, USA) and GAPDH (1:3000, Proteintech, 10494-1-AP, Chicago, IL, USA).

Techniques: Expressing, Quantitative Proteomics, Over Expression, Immunohistochemical staining, Staining, Western Blot, Quantitative RT-PCR, Standard Deviation

Journal: Oncology Research

Article Title: Single-Cell and Multi-Omics-Based Characterization of Gastric Cancer Identifies TPP1 as a Potential Target for Gastric Cancer Progression and Treatment

doi: 10.32604/or.2026.070208

Figure Lengend Snippet: Silencing of TPP1 attenuates tumorigenic properties and enhances apoptosis in gastric cancer cells. ( A ) qRT-PCR analysis confirmed elevated TPP1 expression in gastric cancer cell lines (n = 3). ( B ) Western blot analysis validated high TPP1 protein levels across gastric cancer cell lines (n = 3). ( C ) qRT-PCR confirmed effective knockdown of TPP1 following siRNA transfection (n = 3). ( D ) Colony formation assays demonstrated a significant reduction in clonogenic capacity upon TPP1 silencing (n = 3). ( E ) Flow cytometry analysis using Annexin V/DAPI staining revealed increased apoptosis in TPP1-depleted cells (n = 3). ( F ) Transwell invasion assays showed a marked decrease in invasive capacity following TPP1 knockdown (n = 3). ( G ) CCK-8 assays indicated impaired cell viability in TPP1-silenced gastric cancer cells (n = 3). Data are presented as mean ± standard deviation (SD), and all experiments were performed in triplicate. Statistical significance was assessed using Student’s t-test unless otherwise specified: p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), p < 0.0001 (****).

Article Snippet: After blocking at room temperature for 1 h, membranes were incubated overnight at 4°C with primary antibodies against TPP1 (1:3000, Proteintech, 25849-1-AP, Chicago, IL, USA) and GAPDH (1:3000, Proteintech, 10494-1-AP, Chicago, IL, USA).

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Knockdown, Transfection, Flow Cytometry, Staining, CCK-8 Assay, Standard Deviation

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: Briefly, biotinylated monoclonal anti-TPP1 antibody (R&D Systems no. MAB2237) was added to a streptavidin-coated MSD plate that has been blocked before use.

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: Briefly, biotinylated monoclonal anti-TPP1 antibody (R&D Systems no. MAB2237) was added to a streptavidin-coated MSD plate that has been blocked before use.

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: Briefly, biotinylated monoclonal anti-TPP1 antibody (R&D Systems no. MAB2237) was added to a streptavidin-coated MSD plate that has been blocked before use.

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: Briefly, biotinylated monoclonal anti-TPP1 antibody (R&D Systems no. MAB2237) was added to a streptavidin-coated MSD plate that has been blocked before use.

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: Briefly, biotinylated monoclonal anti-TPP1 antibody (R&D Systems no. MAB2237) was added to a streptavidin-coated MSD plate that has been blocked before use.

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: Briefly, biotinylated monoclonal anti-TPP1 antibody (R&D Systems no. MAB2237) was added to a streptavidin-coated MSD plate that has been blocked before use.

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: Briefly, biotinylated monoclonal anti-TPP1 antibody (R&D Systems no. MAB2237) was added to a streptavidin-coated MSD plate that has been blocked before use.

Techniques: Immunostaining, Expressing, Electrochemiluminescence, Concentration Assay

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