Journal: European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences

Article Title: Pyrrolidine SS13 Induces Oxidative Stress and Autophagy-Mediated Cell Death in Colorectal Cancer Cells.

doi: 10.1016/j.ejps.2024.106982

Figure Lengend Snippet: Figure 11. Immunofluorescence staining of LAMP1 and LysoTracker in HCT116 (A) and Caco-

Article Snippet: LAMP1 (D2D11) XP® Rabbit mAb (Cell Signaling Technology, Danvers, MA, USA) and LC3B (E5Q2K) Mouse mAb (Cell Signaling Technology, Danvers, MA, USA) primary antibodies were used on the cells for 90 min at room temperature.

Techniques: Immunofluorescence, Staining

Journal: European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences

Article Title: Pyrrolidine SS13 Induces Oxidative Stress and Autophagy-Mediated Cell Death in Colorectal Cancer Cells.

doi: 10.1016/j.ejps.2024.106982

Figure Lengend Snippet: Figure 14. Immunofluorescence staining of LAMP1 and LC3B in HCT116 after SS13 (7 µM),

Article Snippet: LAMP1 (D2D11) XP® Rabbit mAb (Cell Signaling Technology, Danvers, MA, USA) and LC3B (E5Q2K) Mouse mAb (Cell Signaling Technology, Danvers, MA, USA) primary antibodies were used on the cells for 90 min at room temperature.

Techniques: Immunofluorescence, Staining

Journal: European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences

Article Title: Pyrrolidine SS13 Induces Oxidative Stress and Autophagy-Mediated Cell Death in Colorectal Cancer Cells.

doi: 10.1016/j.ejps.2024.106982

Figure Lengend Snippet: Figure 15. Immunofluorescence staining of LAMP1 and LC3B in Caco-2 cells treated with SS13

Article Snippet: LAMP1 (D2D11) XP® Rabbit mAb (Cell Signaling Technology, Danvers, MA, USA) and LC3B (E5Q2K) Mouse mAb (Cell Signaling Technology, Danvers, MA, USA) primary antibodies were used on the cells for 90 min at room temperature.

Techniques: Immunofluorescence, Staining

Journal: Nature Communications

Article Title: A pathogenic Tau mutation drives autophagy-lysosome dysfunction that limits Tau degradation in a model of frontotemporal dementia

doi: 10.1038/s41467-026-70473-5

Figure Lengend Snippet: A iPSC-derived NGN2 neurons (control) were cultured until DIV14 and immuno-stained for LAMP1+ vesicles (green), MAP2 (magenta). Scale bar 5 µm. Right inset: LAMP1+ vesicles (Red) co-stained for total Tau (Tau5; green) and pTau (AT180; green). Imaging by Airyscan super-resolution microscopy. Scale bar 0.5 µm. B Tau and pTau localization within LAMP1+ vesicles differ. Dashed line used as a reference for subsequent quantification. LAMP1+ vesicles (red) co-stained for total Tau (Tau5; green) and pTau (AT180; green). Scale bar 0.5 µm. C , D Quantification of fluorescent intensity of Tau (Tau5; C ) and pTau (AT180; D ) in LAMP1+ vesicles across the dashed line in ( B ). The data displayed is a representation of the mean localization calculated across 3 independent experiments. See Supplementary Fig. for individual traces. C Tau, n = 35 LAMP1+ vesicles. D pTau, n = 27 LAMP1+ vesicles. E Quantification of the percentage of LAMP1+ vesicles per cell that contain total Tau or pTau (Tau+ or free of Tau, Tau-). Graph represents mean ± SEM. 186 LAMP1+ vesicles were quantified from three independent experiments. Two-tailed Mann–Whitney U- test. *** p = 0.0043. F Immunogold electron microscopy of pTau in iPSC-derived NGN2 neurons. Immunogold labeling with the anti-pTau antibody (AT180) is associated with the single-membrane vesicles containing electron-dense material (defined as lysosomes). Mitochondria (Mito), the endoplasmic reticulum (ER), and the nucleus (Nuc) are visible but remain unlabeled. pTau predominantly localized to the membranes of electron-dense structures with single-limiting membranes (lysosomes, right inset). Additionally, some structural labeling was observed along microtubules. Scale bar 1 µm (left) and 50 nm (right, inset). G Quantification of gold particle-labeled pTau localization within lysosomal structures. The graph represents mean ± SEM. 26 lysosomes were quantified. Two-tailed Mann–Whitney U -test. **** p < 0.0001. H Diagram of Tau and pTau localization in lysosomes based on our observations. Source data are provided as a Source Data file. Created in BioRender. Mirfakhar, F. (2026) https://BioRender.com/8bzn5l2 .

Article Snippet: Lysosome characterization analysis was performed using LAMP1 antibody (D2D11; Cell Signaling, 9091) on 60x confocal images acquired in Z-stacks.

Techniques: Derivative Assay, Control, Cell Culture, Staining, Imaging, Super-Resolution Microscopy, Two Tailed Test, MANN-WHITNEY, Electron Microscopy, Labeling, Membrane

Journal: Nature Communications

Article Title: A pathogenic Tau mutation drives autophagy-lysosome dysfunction that limits Tau degradation in a model of frontotemporal dementia

doi: 10.1038/s41467-026-70473-5

Figure Lengend Snippet: A , C MAPT WT and p.R406W neurons were stained for LAMP1 (red) and MAP2 (green). Scale bar 10 µm. B Magnified images of LAMP1+ vesicles (red) and total Tau (Tau5; green). Representative images of LAMP1+ vesicles free of total Tau (top panel), total Tau staining on the lysosomal membrane (middle panel), and total Tau staining in the lysosomal lumen (bottom panel). Scale bar 1 µm. D Magnified images of LAMP1+ vesicles (red) and pTau (AT180; green). Representative images of LAMP1+ vesicles free of pTau (top panel), pTau detected on the lysosomal membrane (middle panel), and pTau detected in the lysosomal lumen (bottom panel). Scale bar 1 µm. E , F Quantification of the percentage of LAMP1+ vesicles per cell. 186 LAMP1+ vesicles were quantified. Two-way ANOVA. E. LAMP1+ vesicles empty of total Tau or with total Tau in the membrane vs lumen. Total Tau; WT: Empty vs. p.R406W: Empty, ** p = 0.002; WT: Membrane vs. p.R406W: Membrane, * p = 0.017; WT: Lumen vs. p.R406W: Lumen, ** p = 0.002. % of lysosomes: Total Tau; WT: Empty = 68.75%; Membrane = 11.84%; Lumen = 19.41%. Total Tau; p.R406W: Empty = 3.65%; Membrane = 26.74%; Lumen = 69.61%. F. LAMP1+ vesicles empty of pTau or with pTau in the membrane vs lumen. pTau, WT: Empty vs. p.R406W: Empty, * p = 0.015; WT: Membrane vs. p.R406W: Membrane, * p = 0.016; WT: Lumen vs. p.R406W: Lumen p = 0.73. % of lysosomes: pTau; WT: Empty = 51.23%; Membrane = 26.27%; Lumen = 22.5%. pTau; p.R406W: Empty = 10.34%; Membrane = 70.3%; Lumen = 19.36%. G Immunogold labeling with the anti-pTau antibody (AT180) in MAPT WT and p.R406W neurons is associated with the single-membrane vesicles containing electron-dense material (defined as lysosomes). Scale bar 0.5 µm. H Quantification of gold particle-labeled pTau localization within lysosomal structures. 53 lysosomes were quantified (WT n = 26; p.R406W n = 27). Kruskal–Wallis test followed by Dunn’s test; ** p = 0.0085; *** p = 0.0005; **** p < 0.0001; ns, not significant. Data represent mean ± SEM. Data representative of 3 independent experiments. Source data are provided as a Source Data file.

Article Snippet: Lysosome characterization analysis was performed using LAMP1 antibody (D2D11; Cell Signaling, 9091) on 60x confocal images acquired in Z-stacks.

Techniques: Staining, Membrane, Labeling

Journal: Nature Communications

Article Title: A pathogenic Tau mutation drives autophagy-lysosome dysfunction that limits Tau degradation in a model of frontotemporal dementia

doi: 10.1038/s41467-026-70473-5

Figure Lengend Snippet: A Differentially expressed genes in MAPT p.R406W neurons are enriched in pathways associated with the autophagy-lysosome pathway (FDR \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\le$$\end{document} ≤ 0.05). White bars, pathways with genes significantly upregulated in MAPT p.R406W neurons. Gray bars, pathways with genes significantly downregulated in MAPT p.R406W neurons. B Schematic. LSD, lysosomal storage disease. C Representative Tuj1+ (green) neurons at DIV14, stained for LAMP1 (red). Right, magnified images of LAMP1+ donut-shaped structures (red) distributed in neurons. Scale bar 5 µm. Inset, scale bar 0.5 µm. Images acquired from 3 independent experiments. D Tuj1+ (green) neurons at DIV14. MAPT p.R406W or isogenic controls (WT) were stained for LAMP1 (red). White arrows, lysosomes in neurites. Scale bar 5 µm. E – G Lysosomal morphology quantified in MAPT p.R406W neurons compared with isogenic controls (WT). Data are mean ± SEM from 3 independent experiments. Data normalized to WT. Two-tailed Mann-Whitney U test. E Quantification of LAMP1+ vesicle density defined as the total number of LAMP1+ puncta within Tuj1+ cells. 19 cells were quantified per genotype. ** p = 0.0048. F LAMP1+ vesicle volume. Lysosomes quantified: MAPT WT n = 208; MAPT p.R406W n = 318. **** p < 0.0001. G Lysosome distance from the nucleus is defined as the shortest distance of LAMP1+ puncta to DAPI + . Lysosomes quantified: MAPT WT n = 164; MAPT p.R406W n = 214. **** p < 0.0001. H iPSC-derived NGN2 neurons (DIV14) were MAP2+ and co-stained with LAMP1 and Cathepsin (CatD). LAMP1+ vesicles (red) co-label with CatD (green) in MAPT p.R406W neurons and isogenic control cells (WT). Scale bar 10 µm. Inset, scale bar 0.5 µm. I Quantification of the percentage of LAMP1 + /CatD+ lysosomes in MAPT WT ( n = 22) and MAPT p.R406W ( n = 16) cells. J Quantification of the percentage of CatD+ vesicles in MAPT WT ( n = 22) and MAPT p.R406W ( n = 16) neurons expressed relative to WT control. p = 0.0373. K Quantification of LAMP1 + /CatD+ size (area). MAPT WT n = 219; MAPT p.R406W n = 242 lysosomes. **** p < 0.0001. Two - tailed Mann–Whitney U -test. 3D reconstruction of Airyscan microscopy images and analyses were performed by Imaris. L Diagram of hypothesized disrupted pathways derived from GO data. Source data are provided as a Source Data file. Created in BioRender. Mirfakhar, F. (2026) https://BioRender.com/8bzn5l2 .

Article Snippet: Lysosome characterization analysis was performed using LAMP1 antibody (D2D11; Cell Signaling, 9091) on 60x confocal images acquired in Z-stacks.

Techniques: Staining, Two Tailed Test, MANN-WHITNEY, Derivative Assay, Control, Microscopy

Journal: Nature Communications

Article Title: A pathogenic Tau mutation drives autophagy-lysosome dysfunction that limits Tau degradation in a model of frontotemporal dementia

doi: 10.1038/s41467-026-70473-5

Figure Lengend Snippet: MAPT p.R406W and isogenic control neurons were treated with G2-567 (0.5 µM) or DMSO for 14 days beginning on DIV7 and were fixed on DIV21. A Neurons (MAP2, green) stained for pTau (AT180, red). Scale bar 20 µm. B Quantification of pTau mean intensity per cell (WT n = 52; WT-G2-567 n = 52; p.R406W n = 59; p.R406W-G2-567 n = 56). Data are represented as mean ± SEM. Data on the y -axis are normalized to WT-DMSO. Kruskal–Wallis test followed by Dunn’s test. **** p < 0.0001. C Neurons (MAP2, green) stained for total Tau (Tau5, magenta). Scale bar 20 µm. D Quantification of Tau mean intensity per cell (WT n = 43; WT-G2-567 n = 63; p.R406W n = 51; p.R406W-G2-567 n = 37). Data are represented as mean ± SEM. Data on the y -axis are normalized to WT-DMSO. Kruskal–Wallis test followed by Dunn’s test. * p = 0.0491 and p = 0.0506. E Quantification of ratio of pTau/total Tau (WT n = 43; WT-G2-567 n = 63; p.R406W n = 51; p.R406W-G2-567 n = 37). Data are represented as mean ± SEM. Data on the y -axis are normalized to WT-DMSO. Kruskal–Wallis test followed by Dunn’s test. ** p = 0.0097. A – E Cells were quantified from 3 independent experiments. F LAMP1+ vesicles (red) co-stained with total Tau (Tau5, green) in MAPT p.R406W and isogenic control neurons after treatment with G2-567. Scale bar 1 µm. G Quantification of the percentage of lysosomes free of total Tau (empty, white) or co-localized with total Tau (Tau positive, gray). Data are represented as mean ± SEM. Tukey’s multiple comparison. Empty vs. Tau positive (+): * p = 0.0355, p = 0.0498, p = 0.0125; *** p = 0.0005; **** p < 0.0001. H LAMP1+ vesicles (red) in MAPT p.R406W and isogenic control neurons after treatment with G2-567. Scale bar 1 µm. I Quantification of lysosome volume (WT n = 135; WT-G2-567 n = 158; p.R406W n = 221; p.R406W-G2-567 n = 177). Data are represented as mean ± SEM. Kruskal-Wallis test followed by Dunn’s test. * p = 0.0109; ** p = 0.0036; **** p < 0.0001. F – I Data are representative of 3 independent experiments. Source data are provided as a Source Data file.

Article Snippet: Lysosome characterization analysis was performed using LAMP1 antibody (D2D11; Cell Signaling, 9091) on 60x confocal images acquired in Z-stacks.

Techniques: Control, Staining, Comparison

Journal: Nature Communications

Article Title: A pathogenic Tau mutation drives autophagy-lysosome dysfunction that limits Tau degradation in a model of frontotemporal dementia

doi: 10.1038/s41467-026-70473-5

Figure Lengend Snippet: MAPT p.R406W and isogenic control (WT) neurons were treated with G2-567 (0.5 µM) or DMSO for 14 days beginning on DIV7 and fixed on DIV21. Neurons were then immunostained for lysosomal positioning and autophagy markers. A Immunostaining for MAP2 (green) and LAMP1 (red). Lower panel, 3D reconstruction by Imaris. Scale bar 5 µm. B Quantification of lysosome density: WT ( n = 16), WT-G2-567 ( n = 14), p.R406W ( n = 17), p.R406W-G2-567 ( n = 19). Kruskal-Wallis test followed by Dunn’s test. * p = 0.0214, p = 0.0390; ** p = 0.0017. C Quantification of lysosome distance from the nucleus as the shortest distance of LAMP1+ vesicles from DAPI. Quantification performed by Imaris 3D rendering. Data on the y -axis are normalized to WT-DMSO. Number of vesicles quantified: WT ( n = 181), WT-G2-567 ( n = 219), p.R406W ( n = 319), p.R406W-G2-567 ( n = 318). Kruskal–Wallis test followed by Dunn’s test. WT-DMSO vs. p.R406W-DMSO: ** p = 0.0011. p.R406W-DMSO vs. p.R406W-G2-567: p = 0.3235. D Immunostaining with MAP2+ (magenta) and JIP3 (green). Scale bar 10 µm. E Quantification of JIP3 mean intensity per cell. WT (n = 43), WT-G2-567 ( n = 55), p.R406W ( n = 33), p.R406W-G2-567 ( n = 54) cells. Kruskal–Wallis test followed by Dunn’s test; **** p < 0.0001. F Cells were probed for autophagosomes using CYTO-ID (green) and microtubule (magenta). Right panel, magnified CYTO-ID positive vesicles (green). Scale bar 5 µm. G Quantification of CYTO-ID mean density. Total number of cells quantified: WT ( n = 71), WT-G2-567 ( n = 79), p.R406W ( n = 79), p.R406W-G2-567 ( n = 78). Kruskal–Wallis test followed by Dunn’s test, ** p = 0.0027; **** p < 0.0001. H Immunostaining for MAP2 (magenta), LC3B (red) and p62 (green). Scale bar 10 µm. I Quantification of LC3B mean intensity per cell. WT ( n = 70), WT-G2-567 ( n = 43), p.R406W ( n = 68), p.R406W-G2-567 ( n = 68) cells. Kruskal-Wallis test followed by Dunn’s test, * p = 0.0393; ** p = 0.0062; *** p = 0.0005; **** p < 0.0001. J Quantification of p62 mean intensity. WT ( n = 26), WT-G2-567 ( n = 32), p.R406W ( n = 32), p.R406W-G2-567 ( n = 26) cells. Kruskal–Wallis test. * p = 0.0266, **** p < 0.0001. All data are mean ± SEM. All data are normalized to WT-DMSO. Data are representative of 3 independent experiments except in panels I and J which represent 4 independent experiments. Source data are provided as a Source Data file.

Article Snippet: Lysosome characterization analysis was performed using LAMP1 antibody (D2D11; Cell Signaling, 9091) on 60x confocal images acquired in Z-stacks.

Techniques: Control, Immunostaining

Journal: iScience

Article Title: De novo peptide grafting to a self-assembling nanocapsule yields a hepatocyte growth factor receptor agonist

doi: 10.1016/j.isci.2021.103302

Figure Lengend Snippet:

Article Snippet: Cells were permeabilized and blocked with 0.05% Triton X-100, 1% BSA in PBS, and stained with 1 μg/ml anti-His-tag mouse monoclonal antibody (OGHis, Medical & Biological Laboratories) and anti-EEA1 rabbit monoclonal antibody (endosomal marker, C45B10, Cell Signaling Technology, 1:200 dilution) or anti-LAMP1 rabbit monoclonal antibody (lysosomal marker, D2D11, Cell Signaling Technology, 1:200 dilution), followed by 1 μg/ml Alexa Fluor 488 conjugated-anti-mouse IgG antibody (Thermo Fisher Scientific) and 1 μg/ml Alexa Fluor 594-conjugated anti-rabbit IgG antibody (Thermo Fisher Scientific).

Techniques: Virus, Recombinant, Expressing, Software

Journal: Science translational medicine

Article Title: Adeno-associated viral vector serotype 9-based gene therapy for Niemann-Pick disease type A

doi: 10.1126/scitranslmed.aat3738

Figure Lengend Snippet: (A and B) Representative images of ASM protein expression in the cortex and cerebellum of NHP after CM delivery of 6 ml of 2.3 × 1013 VG/ml AAV9-hASM throughout CM injection at 1-month (A) and 3-month (B) survival time (n = 1 animal per group). Insets depict the intracellular lysosomal/endosomal distribution pattern of the hASM protein in the cells indicated by black arrowheads. (C) ASM protein expression in the cortex and cerebellum of control NHP not injected with AAV9-hASM. (D) Immunofluorescent colocalization of ASM and the lysosomal marker Lamp1 in Purkinje cells of the cerebellum. (E) Graphs showing the mean ± SEM of ASM-associated infrared signal per area in the indicated brain regions of noninjected NHP (white bars), 1-month survival NHP (black bars), and 3-month survival NHP (gray bars) after CM AAV9-hASM injection (n = 1 animal per group). (F) Graph showing mean ± SEM ASM-associated infrared signal per area in the thalamus of noninjected NHP (white bar), 1-month survival NHP (black bar), and 3-month survival NHP (gray bar) after CM AAV9-hASM injection (n = 1 animal per group). (G) Graph showing ASM amount in liver homogenates determined by ELISA in noninjected NHP (white bar), 1-month survival NHP (black bar), and 3-month survival NHP (gray bar) after CM AAV9-hASM injection (n = 1 animal per group). (H) Graphs showing ASM amount determined by ELISA in representative regions (cervical, thoracic, and lumbar) of the spinal cord of noninjected NHP (white bars) or in NHP 1 month (black bars) and NHP 3 months (gray bars) after CM AAV9-hASM injection (n = 1 animal per group). (I) Representative images of H&E staining in cortex and cerebellum of NHP after CM delivery of AAV9-hASM throughout CM injection at 1- and 3-month survival time and in a noninjected control NHP. (J) Representative images of Purkinje cells within the cerebellum stained against calbindin in NHP after CM delivery of AAV9-hASM at 1- and 3-month survival time and in a noninjected control NHP. Scale bars, 50 μm for (A), (B), and (C) insets; 500 μm for (A) and (B) (top left); 200 μm for (A) and (B) (bottom left and top right), (C), (I), and (J); 100 μm for (A) and (B) (bottom right); 10 μm for (D). N, nucleus; a.u., arbitrary units. In (H), the symbol ⌘ in the control animal denotes levels of ASM lower than the detection limit of the ELISA kit. Statistical analyses were performed using two-way ANOVA followed by Tukey’s post hoc test for multiple comparisons correction. *P < 0.05, **P < 0.01, and ***P < 0.001. N/A, not available.

Article Snippet: Lysosomal/endosomal distribution pattern was assessed with double-label immunofluorescence for ASM (mouse anti-hASM, 1:100, Sanofi) and a lysosomal marker, LAMP-1 (rabbit anti-LAMP1, 1:200, D2D11, Cell Signaling Technologies).

Techniques: Expressing, Injection, Marker, Enzyme-linked Immunosorbent Assay, Staining

Journal: Science translational medicine

Article Title: Adeno-associated viral vector serotype 9-based gene therapy for Niemann-Pick disease type A

doi: 10.1126/scitranslmed.aat3738

Figure Lengend Snippet: (A) Immunofluorescence images of hASM expression in Purkinje cells (indicated by asterisks) after iCb and CM injection of AAV9-hASM. ML, molecular layer; PCL, Purkinje cell layer; and GL, granular layer. (B) Phenotypic analysis of AAV9-hASM transduced cells in the cerebellum of iCb and CM AAV9-hASM-injected ASM-KO mice. Brain sections were costained in green against hASM and in red against neuronal (MAP2), astrocytic (GFAP), or microglia (Iba1) markers. Asterisks depict examples of cells coexpressing hASM and the cell type-specific marker. (C) Western blot analysis of hASM expression in cerebellar homogenates from ASM-KO mice injected with aCSF and iCb or CM AAV9-hASM. Graph shows mean ± SEM cerebellar hASM amount normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (n = 4 per group). One-way ANOVA followed by Bonferroni post hoc. (D) Mean ± SEM SM concentration in cerebellar homogenates from WT and ASM-KO mice after receiving iCb or CM injection of aCSF or AAV9-hASM (n = 3 to 6 per group). Two-way ANOVA followed by Bonferroni (iCb) and Games-Howell (CM) post hoc. (E) Immunofluorescence staining against lysosomal Lamp1 in Purkinje cells (depicted by white dot lines) after aCSF or AAV9-hASM in iCb or CM injection in WT and ASM-KO mice. Graphs show mean ± SEM size of lysosomes in Purkinje cells after aCSF or AAV9-hASM iCb or CM injection in WT and ASM-KO mice (n = 3 to 6 per group). Two-way ANOVA followed by Bonferroni post hoc. (F) Purkinje cell density analysis by immunofluorescent staining of calbindin in the posterior lobe of the cerebellum after aCSF or AAV9-hASM iCb or CM injections in WT and ASM-KO mice. Graphs show mean ± SEM number of Purkinje cells after aCSF or AAV9-hASM iCb or CM injections in WT and ASM-KO mice (n = 3 to 6 per group). Two-way ANOVA followed by Bonferroni (iCb) and Games-Howell (CM) post hoc. DAPI (4′,6-diamidino-2-phenylindole) staining in blue identifies the cell nuclei in the different panels. Scale bars, 50 μm for (A) and (B); 10 μm for (E); and 100 μm for (F). *P < 0.05, **P < 0.005, and ***P < 0.001.

Article Snippet: Lysosomal/endosomal distribution pattern was assessed with double-label immunofluorescence for ASM (mouse anti-hASM, 1:100, Sanofi) and a lysosomal marker, LAMP-1 (rabbit anti-LAMP1, 1:200, D2D11, Cell Signaling Technologies).

Techniques: Immunofluorescence, Expressing, Injection, Marker, Western Blot, Concentration Assay, Staining