mouse tissue sections  (Cell Signaling Technology Inc)

Journal: Cell reports

Article Title: Granulins rescue inflammation, lysosome dysfunction, lipofuscin, and neuropathology in a mouse model of progranulin deficiency

doi: 10.1016/j.celrep.2024.114985

Figure Lengend Snippet: (A) Constructs including coding region, domains, and epitope tags that were packaged into rAAV2/1 (Twin-Strep tag; V5 tag; FLAG tag; SP, signal peptide; paragranulin; granulin-1 [GRN1; G]; GRN2 [F]; GRN3 [B]; GRN4 [A]; GRN5 [C]; GRN6 [D]; GRN7 [E]). (B) Experimental workflow includes intracerebroventricular (i.c.v.) injection of rAAV, mouse aging, sample collection, and sample analysis. (C) ELISA quantification of hPGRN in cortical tissue from rAAV-injected mice as mean ± SD. One-way ANOVA with Tukey’s post hoc correction. n = 6–7 mice/group. * p < 0.05 and ** p < 0.01. (D) Immunoblot of cortical and hippocampal lysates verifying expression of GFP, hGRN2, and hGRN4 (β-tubulin loading control). (E) IHC images of Twin-Strep to visualize expression of GFP, hPGRN, hGRN2, and hGRN4 in coronal section plus magnified images of the cortex, hippocampus, and thalamus. Scale bars: 2 μm (full section) and 200 μm (magnified boxes). (F) Immunofluorescence (IF) images co-staining for hPGRN, hGRN2, and hGRN4 and antibody markers for neurons (Map2) and microglia (Iba1) in the cortex of an hPGRN, hGRN2- Grn −/− , and hGRN4- Grn −/− mouse. Scale bar: 10 μm.

Article Snippet: Three-dimensional visualization and analysis of colocalization between lysosomal marker, CTSD and granulins in hPGRN, hGRN2, and hGRN4 injected mouse brain tissue sections, we employed IMARIS (v.10.0, Bitplane).

Techniques: Construct, Strep-tag, FLAG-tag, Injection, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Control, Immunofluorescence, Staining

Journal: Cell reports

Article Title: Granulins rescue inflammation, lysosome dysfunction, lipofuscin, and neuropathology in a mouse model of progranulin deficiency

doi: 10.1016/j.celrep.2024.114985

Figure Lengend Snippet: (A) Volcano plot of upregulated (yellow) and downregulated proteins (blue) in the thalamus of GFP- Grn −/− vs. GFP- Grn +/+ mice (fold change [FC] > 1.2, p < 0.05). (B) Bar graph of the most significantly enriched Gene Ontology (GO) terms describing the differentially expressed proteins in (A) (GFP- Grn −/− mice vs. GFP- Grn +/+ mice; FC = 1.2 and adjusted p value = 0.05). Displaying all significant changed modules ( p < 0.05). (C) Plot of PCs (PC1 vs. PC2) for GFP- Grn +/+ mice (blue), GFP- Grn −/− mice (gray), hPGRN- Grn −/− mice (purple), hGRN2- Grn −/− mice (green), and hGRN4-GFP- Grn −/− mice (yellow). Ellipses: 95% confidence interval. (D) Heatmap of top 140 proteins (rows) differentially expressed between GFP- Grn −/− and GFP- Grn +/+ and treatment groups (columns). Quantification of individual proteins shown (log 2 Z score transformed). Individual mouse numbers are below the column. (E) Bar plots comparing correction of elevated levels of LGALS3, CD68, GFAP, GPNMB, HEXB, LYZ2, MPEG1, SERPINA3N, and TPP1 in Grn −/− mice injected with GFP, hGRN2, hGRN4, or hPGRN. Mean (protein abundance) ± SD. One-way ANOVA with Tukey’s post hoc. n = 5–7 mice/group. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. (F) Correlation of hGRN2 (green) and hGRN4 (yellow) expression with galectin-3 (R = 0.78, p = 0.0011). (G) Correlation of hGRN2 (green) and hGRN4 (yellow) expression with P2RY12 (R = 0.77, p = 0.0014).

Article Snippet: Three-dimensional visualization and analysis of colocalization between lysosomal marker, CTSD and granulins in hPGRN, hGRN2, and hGRN4 injected mouse brain tissue sections, we employed IMARIS (v.10.0, Bitplane).

Techniques: Transformation Assay, Injection, Expressing

Journal: Cell reports

Article Title: Granulins rescue inflammation, lysosome dysfunction, lipofuscin, and neuropathology in a mouse model of progranulin deficiency

doi: 10.1016/j.celrep.2024.114985

Figure Lengend Snippet: (A) Heatmap of differentially expressed (log 2 Z score transformed) lysosomal proteins (GO module) in GFP- Grn −/− and GFP- Grn +/+ mice. 42 proteins are included (rows) across mice from all treatment groups (columns). (B and C) Representative (B) cathepsin Z and (C) galectin-3 IHC of coronal sections from rAAV-injected groups (GFP, hPGRN, hGRN2, hGRN4). Scale bar: 2 mm. (D) Quantification of cathepsin Z IHC signal in cortex, hippocampus, and thalamus. (E) Immunoblot for cathepsin Z in cortical, hippocampal, and thalamic brain lysates from all injection groups. (F–H) Quantification of (F) cortical, (G) thalamic, and (H) hippocampal immunoblot of cathepsin Z normalized to H3. (I) Quantification of galectin-3 IHC signal in cortex, hippocampus, and thalamus. (J) Immunoblot for galectin-3 in cortical, hippocampal, and thalamic brain lysates from all injection groups. (K–M) Quantification of (K) cortical, (L) thalamic, and (M) hippocampal galectin-3 immunoblot normalized to H3. Data are presented as means ± SD. n = 5 mice/group. p values were calculated by one-way or two-way (D and I) ANOVA with Tukey’s post hoc analysis. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: Three-dimensional visualization and analysis of colocalization between lysosomal marker, CTSD and granulins in hPGRN, hGRN2, and hGRN4 injected mouse brain tissue sections, we employed IMARIS (v.10.0, Bitplane).

Techniques: Transformation Assay, Injection, Western Blot

Journal: Cell reports

Article Title: Granulins rescue inflammation, lysosome dysfunction, lipofuscin, and neuropathology in a mouse model of progranulin deficiency

doi: 10.1016/j.celrep.2024.114985

Figure Lengend Snippet: (A) Representative images of fluorescent immunohistochemistry of Grn −/− mice injected with AAV-hPGRN, AAV-hGRN2, or AAV-hGRN4 stained for hGRNs (hPGRN, hGRN2, or hGRN4; red), lysosomal protein cathepsin D (CTSD; green), and nucleus (DAPI stain; blue). Images were analyzed with IMARIS software for voxel co-localization (white). Scale bar: 10 μm. (B) Representative images of fluorescent immunocytochemistry of MEF Grn −/− TMEM192 3xHA cells expressing hPGRN, hGRN2, and hGRN4 stained for lysosomal protein CTSD (green), hGRNs (PGRN, GRN2, or GRN4; red), mitochondrial protein heat shock protein 60 (HSP60; gray), and nucleus (DAPI stain; blue). Scale bar: 10 μm. (C) Quantification of Pearson’s correlation coefficients (PCCs) between CTSD and hGRNs vs. HSP60 and hGRNs in MEF Grn −/− TMEM192–3xHA cells expressing hPGRN, hGRN2, or hGRN4. Data are represented as mean ± SD. n = 5 area/group. p values were determined by two-way ANOVA with Tukey’s post hoc analysis. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: Three-dimensional visualization and analysis of colocalization between lysosomal marker, CTSD and granulins in hPGRN, hGRN2, and hGRN4 injected mouse brain tissue sections, we employed IMARIS (v.10.0, Bitplane).

Techniques: Immunohistochemistry, Injection, Staining, Software, Immunocytochemistry, Expressing

Journal: Cell reports

Article Title: Granulins rescue inflammation, lysosome dysfunction, lipofuscin, and neuropathology in a mouse model of progranulin deficiency

doi: 10.1016/j.celrep.2024.114985

Figure Lengend Snippet: (A) Lysosome immunoprecipitation (lyso-IP) workflow using MEF Grn −/− cells co-expressing TMEM192–3xHA and hPGRN, hGRN2, or hGRN4. (B) Immunoblots of cell lysate (cyto), input, and lyso-IP fractions isolated from MEF Grn −/− TMEM192–3xHA cells expressing hPGRN, hGRN2, and hGRN4 probed for lyso-tag (HA), mouse PGRN, hPGRN, hGRN2, hGRN4, lysosome (LAMP1 and CTSZ), mitochondria (HSP60), endoplasmic reticulum (PDI), and cytoskeleton (β-actin).

Article Snippet: Three-dimensional visualization and analysis of colocalization between lysosomal marker, CTSD and granulins in hPGRN, hGRN2, and hGRN4 injected mouse brain tissue sections, we employed IMARIS (v.10.0, Bitplane).

Techniques: Immunoprecipitation, Expressing, Western Blot, Isolation

Journal: Cell reports

Article Title: Granulins rescue inflammation, lysosome dysfunction, lipofuscin, and neuropathology in a mouse model of progranulin deficiency

doi: 10.1016/j.celrep.2024.114985

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Three-dimensional visualization and analysis of colocalization between lysosomal marker, CTSD and granulins in hPGRN, hGRN2, and hGRN4 injected mouse brain tissue sections, we employed IMARIS (v.10.0, Bitplane).

Techniques: Recombinant, Plasmid Preparation, Virus, Staining, Blocking Assay, Hydrophilic Interaction Liquid Chromatography, Transfection, Magnetic Beads, Polymer, Avidin-Biotin Assay, Enzyme-linked Immunosorbent Assay, Software

Journal: Cell reports

Article Title: Granulins rescue inflammation, lysosome dysfunction, lipofuscin, and neuropathology in a mouse model of progranulin deficiency

doi: 10.1016/j.celrep.2024.114985

Figure Lengend Snippet:

Article Snippet: Three-dimensional visualization and analysis of colocalization between lysosomal marker, CTSD and granulins in hPGRN, hGRN2, and hGRN4 injected mouse brain tissue sections, we employed IMARIS (v.10.0, Bitplane).

Techniques: