Journal: bioRxiv

Article Title: Toll-like receptor 9 contributes in microglial activation and lysosomal dysfunction to promote Alzheimer’s disease

doi: 10.64898/2026.01.16.699962

Figure Lengend Snippet: (A) Transcriptional analysis of lysosomal-related genes in WT, APP23 and A9KO microglia. n=3. Heatmap shows Z-Score (for genes). (B) Immunofluorescence images of purified microglia from 10 to 15 months old APP23 and A9KO mice stained for IBA-1 (green), cathepsin C (red), lysosomes (LAMP-1, blue) and nucleus (DAPI, yellow). Scale bar: 5 μm. (C) CTSC and LAMP-1 colocalization expressed as Mander’s coefficient. n=20 cells or more per condition for each experiment were analyzed. n=2. *p<0.05; two-tailed student’s t-test was performed. CTSC: cathepsin C. (D) Immunofluorescence images of purified microglia from 10 to 15 months old WT, APP23 and A9KO mice stimulated or not with 0.5 mM LLOMe for 2 h and stained for microglia (IBA-1, blue), LGALS1 (red) and lysosomes (LAMP-1, green). Scale bar: 5 μm. LLOMe: L-Leucyl-L-Leucine Methyl Ester, LGALS1: galectin 1. NS: non-stimulated. (E) Quantification of the number of LGALS1 + /LAMP-1 + puncta per cell (top) and of the percentage of cells with three or more puncta per field acquired (bottom). n= 20 cells or more per condition for each experiment was analyzed using IMARIS. n=3. NS: non-stimulated. ns: not significative, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, one-way ANOVA was performed with Tukey post-hoc test. 4 Immunofluorescence images of purified microglia from 10 to 15 months old APP23 and A9KO mice incubated with 4 μM of Aβ 1-42 fibrils coupled to HilexaFluor 488 (red) for 1, 2, and 6 hours and stained for microglia (IBA-1 green) and nucleus (DAPI, yellow). Scale bar: 5 μm. (F) Aβ 1-42 relative intracellular fluorescence intensity in APP23 and A9KO microglia. n= 20 cells or more per condition for each experiment was analyzed. n=3. *p<0.05, **p<0.01; one-way ANOVA was performed with Tukey post-hoc test.

Article Snippet: Cells were then incubated overnight at 4°C with a combination of the following primary antibodies diluted in blocking buffer (BB) (1X PBS, 2% BSA): anti-galectin 1 antibody (EPR3205) (Abcam, #ab108389), anti-mouse CD107a (LAMP-1) clone 1D4B (Invitrogen, #14-1071-85), anti-human CD107a (BD Pharmingen, #555789), anti-AIF/Iba1 Antibody (Biotechne #NB100-1028), anti-Iba1 antibody (for immunocytochemistry) (Fujifilm Wako chemicals #019-19741), anti-TFEB antibody (Proteintech, #13372-1-AP) and anti-cathepsin C/DPPI antibody (Biotechne, #AF1034).

Techniques: Immunofluorescence, Purification, Staining, Two Tailed Test, Incubation, Fluorescence

Journal: bioRxiv

Article Title: Toll-like receptor 9 contributes in microglial activation and lysosomal dysfunction to promote Alzheimer’s disease

doi: 10.64898/2026.01.16.699962

Figure Lengend Snippet: (A) Immunofluorescence images of human microglia cell line (HMC3) cultured with CpG-A (2 μg/ml) for 96 hours, pre-incubated or not for 1 h with 0.5 μM of AZP2006, treated or not with 1 mM LLOMe for 2 h and stained for LGALS1 (green), lysosomes (LAMP-1, red) and nucleus (DAPI, blue). Scale bar: 5 μm. Scale bar magnification: 2 μm. (B) Quantification of the number of LGALS1 + /LAMP-1 + puncta per cell (top) and of the percentage of cells with three or more puncta (bottom). n= 20 cells or more per condition for each experiment was analyzed. n=3. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; one-way ANOVA was performed with Tukey post-hoc test. (C) IL-6 production by HMC3 cells pre-incubated or not for 1 h with 0.5 μM or 1 μM of AZP2006, stimulated or not with 1 μg/ml of CpG-A or 100 ng/ml of LPS and measured by ELISA. n=3. ns: not significative, *p<0.05, ****p<0.0001, one-way ANOVA was performed with Tukey post-hoc test. NS: non-stimulated. (D) Immunofluorescence images of the pyramidal layer of the hippocampus (top) and entorhinal cortex region (bottom) in human brain samples from a healthy control (n=3, left) and an AD patient (n=3, right) stained for dsDNA (cyan) and nuclear envelope (Lamin B1, magenta). On the left is displayed whole tissue with regions analyzed highlighted. Scale bar whole region: 1 mm. Scale bar insets: 5 μm. PL: pyramidal layer, EC: entorhinal cortex. (E) Quantification of the percentage of dsDNA area in the nucleus (within Lamin B1, left) or total percentage of extranuclear dsDNA for the field acquired (right) in the PL and in the EC. Each dot represents a quantified image (n=3 for PL and n=4 for EC for each individual). ns: not significative, *p<0.05, ***p<0.001; two-tailed student’s t-test was performed.

Article Snippet: Cells were then incubated overnight at 4°C with a combination of the following primary antibodies diluted in blocking buffer (BB) (1X PBS, 2% BSA): anti-galectin 1 antibody (EPR3205) (Abcam, #ab108389), anti-mouse CD107a (LAMP-1) clone 1D4B (Invitrogen, #14-1071-85), anti-human CD107a (BD Pharmingen, #555789), anti-AIF/Iba1 Antibody (Biotechne #NB100-1028), anti-Iba1 antibody (for immunocytochemistry) (Fujifilm Wako chemicals #019-19741), anti-TFEB antibody (Proteintech, #13372-1-AP) and anti-cathepsin C/DPPI antibody (Biotechne, #AF1034).

Techniques: Immunofluorescence, Cell Culture, Incubation, Staining, Enzyme-linked Immunosorbent Assay, Control, Two Tailed Test

Journal: Frontiers in immunology

Article Title: ELK4 exerts opposite roles in cytokine/chemokine production and degranulation in activated mast cells.

doi: 10.3389/fimmu.2023.1171380

Figure Lengend Snippet: FIGURE 4 Elk4 deficiency promotes degranulation and histamine release. (A) Time course and dose-dependent curve detection of the beta-hexosaminidase releases. BMMCs were sensitized with anti-DNP-IgE (1 mg/ml) overnight, then stimulated with DNP-HSA (10, 100, 1000 ng/ml) for 60min (Left) or stimulated with DNP-HSA (100 ng/ml) for 5min, 15min, 30min and 60min (Right). Bar, mean; error bar, SD; n=4; **p<0.01. (B) The total contents of beta-hexosaminidase in Elk4 WT, HZ and KO BMMCs are shown. Bar, mean; error bar, SD; n=3; *p < 0.05; **p<0.01. (C, D) Degranulation of Elk4 WT, HZ and KO PCMCs was assessed by beta-hexosaminidase release assay. PCMCs were sensitized with anti-DNP-IgE (1 mg/ml) overnight and stimulated with DNP-HSA (100 ng/ml) for 1 hour. The released contents (A) and the total contents (B) of beta-hexosaminidase in Elk4 WT, HZ and KO PCMCs are shown. Bar, mean; error bar, SD; n=2; **p<0.01. (E, F) Degranulation of Elk4 WT, HZ and KO BMMCs (E) and PCMCs (F) was assessed by cell-surface LAMP1 staining using flow cytometry. Both BMMCs and PCMCs were sensitized with anti-DNP-IgE (1 mg/ml) overnight and stimulated with DNP-HSA (100 ng/ml) for 1 hour before LAMP1 staining. Bar, mean; error bar, SD; n=3; *p<0.05.

Article Snippet: Antibodies for flow cytometry were as follows: anti-mouse CD45-violetFluor450 (Multi Sciences, AM04512), anti-mouse CD45-PECY7 (BioLegend, 103114), anti-mouse CD19-FITC (Multi Sciences, AM01901), anti-mouse CD3-PE (BioLegend, 100205), anti-mouse CD11b-Percp-cy5.5 (Multi Sciences, AH011B07), anti-mouse GR-1-PE (Multi Sciences, AM0L604), Lamp1-FITC (ProteinTech, FITC-65050), IL-6-eFluor450 (ebioscience, 48-7061-82), CCL3-PE(ebioscience, 12-7532-82), TNFa-APC(ebioscience, 17-7321-82). b-Hexosaminidase assay BMMCs and PCMCs were sensitized with anti-DNP-IgE (1 mg/ml) overnight.

Techniques: Release Assay, Staining, Cytometry

Journal: PLoS ONE

Article Title: Inhibition of Glioma Cell Lysosome Exocytosis Inhibits Glioma Invasion

doi: 10.1371/journal.pone.0045910

Figure Lengend Snippet: GPN treatment decreased glioma cell surface lysosome number. A. Upper: representative confocal images depicting the astrocyte surface Lamp1 (green) and the total Lamp1 (red). Lower: quantification of the intensity ratio (surface/total). Error bars represent the s.e.m. of 42 images from two independent experiments; ***denotes P<0.001 of C6 compared with astrocytes. (Student's t-test). B. The FACS results of LAMP1 surface staining showing more lysosomes on the surface in both C6 and U251 glioma cells than in astrocytes. GPN treatment decreased LAMP1 surface staining compared with the control group. The X axis shows fluorescence intensity and the Y axis shows cell number. The green curve represents the negative control and the red area represents the LAMP1 surface staining group.

Article Snippet: One group was incubated with 50 μl mouse anti-LAMP1 antibody (Assay Designs, VAM-EN001, 1∶50) at 4°C for 1 h, and the other was incubated with the same amount of PBS.

Techniques: Staining, Control, Fluorescence, Negative Control

Journal: PLoS ONE

Article Title: Inhibition of Glioma Cell Lysosome Exocytosis Inhibits Glioma Invasion

doi: 10.1371/journal.pone.0045910

Figure Lengend Snippet: C6 and U251 cells were immunostained with LAMP1 (green), cathepsin D (red), and Rab27a (blue). Bar charts show the percentage of colocalized puncta (labeled by both types of fluorescent dye). The number associated with each column refers to the number of image fields examined for each condition.

Article Snippet: One group was incubated with 50 μl mouse anti-LAMP1 antibody (Assay Designs, VAM-EN001, 1∶50) at 4°C for 1 h, and the other was incubated with the same amount of PBS.

Techniques: Labeling