Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Human Galectin-1 Improves Sarcolemma Stability and Muscle Vascularization in the mdx Mouse Model of Duchenne Muscular Dystrophy

doi: 10.1016/j.omtm.2019.01.004

Figure Lengend Snippet: Assessment of rHsGal1 Treatments in mdx Mice Relative to PBS Controls (A) The i.p. injection schedule for mdx mice treated with PBS and 5, 20, and 50 mg/kg rHsGal1. (B–D) Body weight (B), activity distance traveled (C), and grip strength (D) were assessed weekly in all treatment groups. (E) Linear regression assessment of the average grip strength over time. (F–H) 10-μm transverse TA muscle cryosections for all treatment groups were histologically assessed for CLN percentage (F) along with minimal Feret’s fiber diameter curves (G) and median (H). (I) Diaphragm muscle from animals of all treatment groups and WT animals were biochemically assessed for collagen content using a hydroxyproline assay. (J and K) Gastrocnemius muscle extracts from animals in all treatment groups were assessed by western blotting, and the levels were quantitated for α7A Integrin (J) and β-Dystroglycan (K). Average ± SEM. Significance: *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Blots were probed using α7A and α7B integrin-specific rabbit polyclonal antibody, β-Dystroglycan H-242 (sc-28535, 1:200), β1D Integrin, utrophin (MANCHO3, 1:50, Developmental Studies Hydridoma Bank [DSHB]), and Galectin-1 (1:100, New England Peptide) and normalized to either glyceraldehyde 3-phosphate dehydrogenase (GAPDH V-18, sc-20357, 1:200) or α-tubulin (DM1A, ab7291, Abnova,1:500).

Techniques: Injection, Activity Assay, Hydroxyproline Assay, Western Blot

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Human Galectin-1 Improves Sarcolemma Stability and Muscle Vascularization in the mdx Mouse Model of Duchenne Muscular Dystrophy

doi: 10.1016/j.omtm.2019.01.004

Figure Lengend Snippet: Assessment of i.v. rHsGal1 Treatment in mdx Mice (A) i.v. rHsGal1 toxicity was assessed using 2.5, 5, 10, 15, and 20 mg/kg rHsGal1 (n = 3/treatment), followed by monitoring for 1 h. (B) The pharmacokinetics of rHsGal1 serum levels were assessed after treatments using 1, 2, and 3 mg/kg rHsGal1. Serum rHsGal1 levels were assessed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) for a human-specific Galectin-1 peptide 15, 120, and 720 min post-injection. (C) A 2.5 mg/kg rHsGal1 i.v. delivery preclinical efficacy study was performed in mdx mice using a weekly injection schedule from 5 to 11 weeks. (D and E) Diaphragms from PBS (n = 6) and 2.5 mg/kg rHsGal1 (n = 7) treated mdx mice were assessed for twitch (D) and tetanus (E) force. (F) Treatment groups were assessed by immunofluorescence (IF) for CLN percentage. (G–J) Gastrocnemius sarcolemma stability protein levels were assessed by western blotting for α7A Integrin (G), α7B Integrin (H), β1D Integrin (I), and β-Dystroglycan (J). Average ± SEM.

Article Snippet: Blots were probed using α7A and α7B integrin-specific rabbit polyclonal antibody, β-Dystroglycan H-242 (sc-28535, 1:200), β1D Integrin, utrophin (MANCHO3, 1:50, Developmental Studies Hydridoma Bank [DSHB]), and Galectin-1 (1:100, New England Peptide) and normalized to either glyceraldehyde 3-phosphate dehydrogenase (GAPDH V-18, sc-20357, 1:200) or α-tubulin (DM1A, ab7291, Abnova,1:500).

Techniques: Drug discovery, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Injection, Immunofluorescence, Western Blot

Journal: BMC Cancer

Article Title: Platelet-activating factor receptor (PAF-R)-dependent pathways control tumour growth and tumour response to chemotherapy

doi: 10.1186/1471-2407-10-200

Figure Lengend Snippet: PAF-receptor antagonist, WEB2170, inhibits melanoma growth and in combination with chemotherapy improves the survival of melanoma-bearing mice . (A) B16F10 melanoma cells (5 × 10 5 ) were injected s.c. into C57BL/6 mice and tumours were measured daily with a caliper. Tumour volume was calculated by the formula: maximum diameter × (minimum diameter) 2 × 0.52. WEB2170 (5 mg/Kg) was given i.p. 30 minutes before the tumour followed by daily injections for 12 days. DTIC (40 μg/animal) was injected i.p. every 3 days after tumour implantation. Data represent the mean ± SEM of tumour volume (n = 5). (B) The Kaplan-Mayer survival curve. For the survival experiments, WEB2170-treatment was given once a day and DTIC every 3 days for 35 days or until the animals died (n = 8-9). Statistical analyses were performed using the log rank test and differences were considered significant at p < 0.05. (*) p < 0.05 compared to PBS group.

Article Snippet: Tumours derived from B16F10 melanoma cells were excised and processed for immunohistochemistry to detect the expression of galectin-3 (1:32, ATCC), PAF-R (1:80; Cayman), COX-2 (1:100; Santa Cruz) and activated caspase-3 (1:600; Cell Signaling).

Techniques: Injection

Journal: BMC Cancer

Article Title: Platelet-activating factor receptor (PAF-R)-dependent pathways control tumour growth and tumour response to chemotherapy

doi: 10.1186/1471-2407-10-200

Figure Lengend Snippet: Combined therapy with PAF-receptor antagonist and dacarbazine targets both tumour and microenvironmental elements . B16F10 melanoma cells (5 × 10 5 ) were injected s.c. into C57BL/6 mice and after 12 days the tumours were excised and processed for immunohistochemistry analysis with antibodies to caspase-3 (A) , COX-2 (B) , CD34 (C) or galectin-3 (D) . Alternatively, spleens of animals of the very same groups and of naïve mice were analysed for the presence of galectin-3-expressing cells in the white pulp (E) . WEB2170 (5 mg/Kg) was given 30 min before the tumour followed by daily injections for 12 days. DTIC (40 μg/animal) was injected i.p. every 3 days after tumour implantation. Morphometric analyses were performed using Eclipse Net software (Nikon). Grids were projected onto tissue sections and the number of grid intersections that overlaid an immunoreactive cell was counted. Results are expressed as the % of area occupied by the cells expressing a given marker (frequency). Data represent the mean ± SEM of positive cells (n = 10). Statistical analyses were performed using ANOVA and SNK (Student Neumans-Keuls test) and differences were considered significant at p < 0.05. ( * ) p < 0.05 comparing treated with non-treated (PBS) groups.

Article Snippet: Tumours derived from B16F10 melanoma cells were excised and processed for immunohistochemistry to detect the expression of galectin-3 (1:32, ATCC), PAF-R (1:80; Cayman), COX-2 (1:100; Santa Cruz) and activated caspase-3 (1:600; Cell Signaling).

Techniques: Injection, Immunohistochemistry, Expressing, Software, Marker

Journal: BMC Cancer

Article Title: Platelet-activating factor receptor (PAF-R)-dependent pathways control tumour growth and tumour response to chemotherapy

doi: 10.1186/1471-2407-10-200

Figure Lengend Snippet: PAF-R is expressed within the tumour microenvironment . (A) The presence of PAF-R was evaluated by FACS analysis in cell suspensions from B16F10-derived tumours (histograms on the left). Examples from either mock (PBS) or dacarbazine (DTIC)-treated animals are illustrated. The histograms shown represent the negative control (ctl) and reactivity with the antibody to PAF-R. Cells from within the tumours stained positively with the antibody to PAF-R. The histogram on the right represents the absence of reactivity for the antibody to PAF-R in B16F10 cells grown in vitro . (B) Immunohistochemistry of B16F10-derived tumours using the same antibody to PAF-R, showing specific reactivity to a few cells infiltrating the tumours, consistent with the notion that only a subpopulation of cells within the tumour microenvironment express the PAF-R (arrow indicates a stained cell).

Article Snippet: Tumours derived from B16F10 melanoma cells were excised and processed for immunohistochemistry to detect the expression of galectin-3 (1:32, ATCC), PAF-R (1:80; Cayman), COX-2 (1:100; Santa Cruz) and activated caspase-3 (1:600; Cell Signaling).

Techniques: Derivative Assay, Negative Control, Staining, In Vitro, Immunohistochemistry

Journal: Journal of Lipid Research

Article Title: Comparison between genetic and pharmaceutical disruption of Ldlr expression for the development of atherosclerosis

doi: 10.1016/j.jlr.2022.100174

Figure Lengend Snippet: Taqman probe accession numbers

Article Snippet: Lgals3 , Mm00802901_m1 , Thermo Fisher Scientific.

Techniques:

Journal: Journal of Lipid Research

Article Title: Comparison between genetic and pharmaceutical disruption of Ldlr expression for the development of atherosclerosis

doi: 10.1016/j.jlr.2022.100174

Figure Lengend Snippet: Liver inflammatory gene expression, triglyceride, and cholesterol. Livers were removed from male and female mice that had been injected once weekly with either saline, Ldlr-ASO, or nothing, with or without Ldlr deficiency. A: Expression of inflammatory genes Il6 , Lgals3 , Cd68 , Saa1 , Saa2 , and Saa3 was assessed. B: Representative immunoblot for Mac2 protein, quantified and normalized to β-actin levels (representative of n = 5–6 mice) (M = marker; top band = 55 kD; and bottom band = 35 kD). C, D: Liver histology from Mac2-stained (C) and H&E-stained sections (D), with Mac2 quantification and assessment of lobular inflammation. The scale bar represents 100 μm. E: Hepatic triglycerides and cholesterol were extracted and quantified. F: Plasma Il6 was measured by ELISA. Data are presented as mean ± SEM, n = 5–15 mice/group. ∗ P < 0.05 from saline; # P < 0.05 from Ldlr-ASO.

Article Snippet: Lgals3 , Mm00802901_m1 , Thermo Fisher Scientific.

Techniques: Gene Expression, Injection, Saline, Expressing, Western Blot, Marker, Staining, Clinical Proteomics, Enzyme-linked Immunosorbent Assay

Journal: Cell Death & Disease

Article Title: Trajectory modeling of endothelial-to-mesenchymal transition reveals galectin-3 as a mediator in pulmonary fibrosis

doi: 10.1038/s41419-021-03603-0

Figure Lengend Snippet: A Schematic diagram showing the functional exploration of C3ar1 and galectin-3 in IPF using lentivirus and galectin-3 interference (shRNA group), respectively. Alternatively, antagonists were used to block the binding affinity of C3a and galectin-3 (antagonist group). B , C Body weight and survival of IPF mice with galectin-3 shRNA lentivirus ( B ) (LV C3ar1 shRNA and LV galectin-3 shRNA, n = 5) or antagonists ( C ) (SB290157: antagonist of C3ar1; GB1107: antagonist of GB1107, n = 6). D , E HE and Masson’s trichrome staining of mouse lung sections following lentivirus or antagonist treatments. Scale bar = 500 μm (global view) or 50 μm (detailed view). F Semiquantitative morphological index/scoring of lung sections. The grade ranges from 1 (normal) to 8 (complete fibrosis). n = 30 for each group; * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: After nasal intubation to inject lentivirus into BLM-induced pulmonary fibrosis mice, fibrosis was alleviated, including a reduction of the weight loss and increase of the survival rate, which reached approximately 40% in the LV C3ar1 shRNA group and LV galectin-3 shRNA group, and was significantly higher than the survival rate of the sham group (LV vehicle shRNA) (Fig. ).

Techniques: Functional Assay, shRNA, Blocking Assay, Binding Assay, Staining

Journal: Journal of Biological Chemistry

Article Title: AMP-activated Protein Kinase α2 Protects against Liver Injury from Metastasized Tumors via Reduced Glucose Deprivation-induced Oxidative Stress

doi: 10.1074/jbc.m113.543447

Figure Lengend Snippet: FIGURE 5. AMPK 2 deficiency enhanced ROS accumulation by decreasing mitophagy, which was related with mTOR signaling pathway. A, liver tissue was harvested 12 days after SL4 injection. Electron microscopy shows the mitophagy in hepatocytes from the tumor-bearing liver. B, the livers were harvested 12 days after SL4 injection, lysed, and subjected to Western blot with antibodies against LC3 and GAPDH. The LC3 II levels decreased in the tumor-bearing livers of AMPK 2/ mice. C and D, the livers were harvested 10 and 13 days after SL4 injection, lysed, and subjected to Western blot with antibodies against AMPK 2, p-mTOR, mTOR, p-4ebp1, 4ebp1, and GAPDH. The phosphorylation level of mTOR and its substrate, 4ebp1, both increased in AMPK 2-de- ficient tumor-bearing livers. E, primary hepatocytes underwent 36 h of glucose starvation, with or without autophagy inhibitor (3-MA) treatment, and were harvested for electron microscopic analysis. The mitophagy was reduced by the 3-MA. F, the lysate from primary hepatocytes was analyzed with LC3 antibody by Western blot, after 3 and 8 h of glucose starvation. AMPK 2 deficiency decreased the level of LC3 II. G, primary hepatocytes were cultured with or without glucose for 36 h. Cells were incubated with Mito Tracker for 20 min, and the autophagosome level was then detected by immunofluorescence of the LC3 antibody. Many autophagosomes colocalized with mitochondria, suggesting mitophagy. AMPK 2 deficiency decreased the colocalization. Scale bar, 10 m. H, localization of ROS and mitochondria were detected by Mito Tracker Red and FITC-CM H2DCFDA. Cells were incubated with Mito Tracker for 20 min, and the fluorescence was recorded 20 min after FITC-CM H2DCFDA was added. A large amount of ROS produced after glucose starvation colocalized with mitochondria. DCF indicates FITC-CM H2DCFDA. Scale bar, 10 m. I and J, primary hepatocytes were cultured with or without glucose or 3-MA for 36 h. Hepatocytes were then trypsinized and oxidized to the FITC-CM H2DCFDA for 20 min and analyzed using a FACSCalibur flow cytometer. The results indicated that 3-MA enhanced ROS production after glucose starvation. d, day(s).

Article Snippet: Antibodies against AMPK 1 and AMPK 2 were from Abcam (Cambridge, MA); antibodies against p-AMPK (threonine 172), p-mTOR, mTOR, p-4ebp1, 4ebp1, and cytokeratin 18 were from Cell Signaling Biotechnology (Danvers, MA); antibodies against Galectin-3 (Mac-2) and GAPDH were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA).

Techniques: Injection, Electron Microscopy, Western Blot, Phospho-proteomics, Cell Culture, Incubation, Immunofluorescence, Fluorescence, Produced, Flow Cytometry

Journal: JCI insight

Article Title: AAV-mediated delivery of secreted acid α-glucosidase with enhanced uptake corrects neuromuscular pathology in Pompe mice.

doi: 10.1172/jci.insight.170199

Figure Lengend Snippet: Figure 1. Systemic gene transfer fails to rescue muscle pathology after short-term treatment at a low vector dose. (A) Experimental design: 3.5-month-old KO mice received a single injection of systemic vector (SYS; n = 5) at a dose of 0.5 × 1013 vg/kg. Age-matched wild-type (WT) and untreat- ed Gaa–/– (KO) mice were used as controls. Muscle samples were collected 1 month (mo) after dosing. (B) Western blot analyses of whole muscle lysates with anti-human GAA antibody. Gapdh was used as a loading control. Graph shows GAA activity in muscle tissues from WT, KO, and SYS-treated KO mice. (C) Glycogen content in muscle tissues across the groups. (D) PAS-stained sections of gastrocnemius muscle; PAS-positive material (small dots) is seen in all fibers from KO mice; some fibers (or parts of a fiber) from SYS-treated KO mice appear normal (asterisks). Bars: 50 μm. (E) Western blot analy- ses of whole muscle lysates with the indicated antibodies. No significant decrease in the levels of lysosomal/autophagosomal markers is seen in treated compared to untreated KO. Statistical significance was determined by 1-way ANOVA and unpaired 2-tailed Student’s t test. Graphs represent mean ± SD. *P < 0.05; **P < 0.01; ****P < 0.0001.

Article Snippet: The following primary antibodies (all diluted 1:1,000) were used: total and phosphorylated AMPK (2535; T172 5831; rabbit monoclonal) and total and nonphosphorylated 4EBP1 (4923; T46 9644, rabbit monoclonal) were from Cell Signaling Technology; LAMP1 (CD107a 553792; rat monoclonal; BD Transduction Laboratories), GAA (ab137068; rabbit monoclonal), SQSTM1/p62 (ab56416; mouse monoclonal), and GAPDH (ab9485; rabbit polyclonal) were from Abcam; and galectin-3 (sc-32790; mouse monoclonal) and LC3B (L7543; rabbit polyclonal) were from Santa Cruz Biotechnology and MilliporeSigma, respectively.

Techniques: Plasmid Preparation, Injection, Western Blot, Control, Activity Assay, Staining

Journal: JCI insight

Article Title: AAV-mediated delivery of secreted acid α-glucosidase with enhanced uptake corrects neuromuscular pathology in Pompe mice.

doi: 10.1172/jci.insight.170199

Figure Lengend Snippet: Figure 3. Systemic gene transfer rescues cardiac and diaphragmatic pathology after short-term treatment at an intermediate dose. (A) Experimental design: 3-month-old KO mice received a single injection of systemic vector (SYS; n = 3) at a dose of 2.5 × 1013 vg/kg. Age-matched wild-type (WT) and untreated Gaa–/– (KO) mice were used as controls. Muscle samples were collected 1 month (mo) after dosing. (B) Western blot analyses of cardiac muscle and diaphragm lysates with anti-human GAA antibody. Gapdh was used as a loading control. (C) GAA activity in the heart and diaphragm far exceeds the WT levels. (D and E) PAS-stained sections of cardiac muscle and the diaphragm; PAS-positive material is abundant in the heart and diaphragm of a KO mouse; the pathology and glycogen content are fully normalized in the heart of SYS-treated KO; inset shows PAS-positive residual glycogen in some cells in the diaphragm despite the treatment. Bars: 50 μm. For inset in E, original magnification, ×2. Statistical significance was determined by 1-way ANOVA. Graphs represent mean ± SD. ****P < 0.0001.

Article Snippet: The following primary antibodies (all diluted 1:1,000) were used: total and phosphorylated AMPK (2535; T172 5831; rabbit monoclonal) and total and nonphosphorylated 4EBP1 (4923; T46 9644, rabbit monoclonal) were from Cell Signaling Technology; LAMP1 (CD107a 553792; rat monoclonal; BD Transduction Laboratories), GAA (ab137068; rabbit monoclonal), SQSTM1/p62 (ab56416; mouse monoclonal), and GAPDH (ab9485; rabbit polyclonal) were from Abcam; and galectin-3 (sc-32790; mouse monoclonal) and LC3B (L7543; rabbit polyclonal) were from Santa Cruz Biotechnology and MilliporeSigma, respectively.

Techniques: Injection, Plasmid Preparation, Western Blot, Control, Activity Assay, Staining

Journal: JCI insight

Article Title: AAV-mediated delivery of secreted acid α-glucosidase with enhanced uptake corrects neuromuscular pathology in Pompe mice.

doi: 10.1172/jci.insight.170199

Figure Lengend Snippet: Figure 5. Muscle glycogen, autophagosomal-lysosomal markers, and mTORC1/AMPK signaling return to near normal after short-term systemic gene transfer at an intermediate vector dose. (A) Experimental design: 3-month-old KO mice received a single injection of systemic (SYS n = 4) or liver-secreted (LS n = 4) vector at a dose of 2.5 × 1013 vg/kg. Age-matched wild-type (WT) and untreated Gaa–/– (KO) mice were used as controls. Muscle samples were collected 1 month (mo) after dosing. (B) Western blot analyses of whole muscle lysates with anti-human GAA antibody. The 110 kDa GAA precursor protein is clearly detectable after LS treatment. Gapdh was used as a loading control. Graph shows GAA activity in muscle tissues from WT, untreated KO, and KO treated with SYS or LS vector. (C) Glycogen content in muscle tissues across the groups. (D and E) Western blot analyses of whole muscle lysates with the indicated antibodies. Statistical significance was determined by 1-way ANOVA. Graphs represent mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: The following primary antibodies (all diluted 1:1,000) were used: total and phosphorylated AMPK (2535; T172 5831; rabbit monoclonal) and total and nonphosphorylated 4EBP1 (4923; T46 9644, rabbit monoclonal) were from Cell Signaling Technology; LAMP1 (CD107a 553792; rat monoclonal; BD Transduction Laboratories), GAA (ab137068; rabbit monoclonal), SQSTM1/p62 (ab56416; mouse monoclonal), and GAPDH (ab9485; rabbit polyclonal) were from Abcam; and galectin-3 (sc-32790; mouse monoclonal) and LC3B (L7543; rabbit polyclonal) were from Santa Cruz Biotechnology and MilliporeSigma, respectively.

Techniques: Plasmid Preparation, Injection, Western Blot, Control, Activity Assay

Journal: JCI insight

Article Title: AAV-mediated delivery of secreted acid α-glucosidase with enhanced uptake corrects neuromuscular pathology in Pompe mice.

doi: 10.1172/jci.insight.170199

Figure Lengend Snippet: Figure 9. Systemic gene transfer reverses muscle pathology in old KO mice. (A) Experimental design: 9-month-old KO mice received a single injection of SYS (n = 8) vector at a dose of 2.5 × 1013 vg/kg. Age-matched (15.5-month-old) wild-type (WT) and 13.5-month-old untreated Gaa–/– (KO) mice were used as con- trols. Muscle samples were collected 7 months after dosing. (B) SYS-treated KO mice appear healthy for their age group; a 13.5-month-old KO mouse shows profound muscle weakness and wasting; see Supplemental Videos 1 and 2. (C) Western blot analyses of whole muscle lysates with anti-human GAA antibody. Gapdh was used as a loading control. Graph shows GAA activity in muscle tissues from WT, untreated KO, and SYS-treated KO mice. (D) Glycogen content in muscle tissues across the groups. (E) PAS-stained sections of gastrocnemius muscle from WT, KO, and SYS-treated mice; muscle fibers from SYS-treated mice appear normal. Bars: 50 μm. (F) Western blot analyses of whole muscle lysates with the indicated antibodies. (G) Immunostaining of single fibers with markers for lysosomes (LAMP1; green), autophagosomes (LC3; red), and nuclei (Hoechst dye; blue); muscle fibers from SYS-treated KO mice are free from autophagic buildup. Bright autofluorescent particles consist of lipofuscin, a typical feature in patients and in a KO model of the disease (87); autophagic buildup in 13.5-month-old untreated KO occupies large portions of the fibers (the image shows projection view produced from 6 consecutive optical sections (Z-stack). Bars: 20 μm. (H) Muscle strength was assessed using grip strength test after treatment. Statistical significance was determined by 1-way ANOVA and unpaired 2-tailed Student’s t test. Data presented as mean ± SD; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: The following primary antibodies (all diluted 1:1,000) were used: total and phosphorylated AMPK (2535; T172 5831; rabbit monoclonal) and total and nonphosphorylated 4EBP1 (4923; T46 9644, rabbit monoclonal) were from Cell Signaling Technology; LAMP1 (CD107a 553792; rat monoclonal; BD Transduction Laboratories), GAA (ab137068; rabbit monoclonal), SQSTM1/p62 (ab56416; mouse monoclonal), and GAPDH (ab9485; rabbit polyclonal) were from Abcam; and galectin-3 (sc-32790; mouse monoclonal) and LC3B (L7543; rabbit polyclonal) were from Santa Cruz Biotechnology and MilliporeSigma, respectively.

Techniques: Injection, Plasmid Preparation, Western Blot, Control, Activity Assay, Staining, Immunostaining, Produced

Journal: JCI insight

Article Title: AAV-mediated delivery of secreted acid α-glucosidase with enhanced uptake corrects neuromuscular pathology in Pompe mice.

doi: 10.1172/jci.insight.170199

Figure Lengend Snippet: Figure 10. Systemic gene transfer restores glycogen accumulation to normal levels in the brain after long-term treatment at an intermediate vector dose. (A) Experimental design. Young (3-month-old) and old (8- to 9-month-old) KO mice received a single injection of SYS (n = 3 young; n = 7 old) or LS (n = 3 young; n = 4 old) vector at a dose of 2.5 × 1013 vg/kg. Age-matched wild-type (WT) and untreated Gaa–/– (KO) mice were used as controls. The samples were collected 7–8 months after dosing. (B) Western blot analyses of whole brain lysates with anti-human GAA antibody. The 110 kDa GAA precursor pro- tein is the predominant form in the brains of LS-treated KO mice. Gapdh was used as a loading control. (C and D) Graphs show GAA activity and glycogen content in brain tissues across the groups in young and old animals. (E) PAS-stained sections of brain tissues (cerebellum and hindbrain) across the groups in old animals; brain sections from SYS-treated mice appear normal; brain sections from LS-treated mice show glycogen storage in neurons and glial cells. Bars: 50 μm. Statistical significance was determined by 1-way ANOVA. Graphs represent mean ± SD. **P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: The following primary antibodies (all diluted 1:1,000) were used: total and phosphorylated AMPK (2535; T172 5831; rabbit monoclonal) and total and nonphosphorylated 4EBP1 (4923; T46 9644, rabbit monoclonal) were from Cell Signaling Technology; LAMP1 (CD107a 553792; rat monoclonal; BD Transduction Laboratories), GAA (ab137068; rabbit monoclonal), SQSTM1/p62 (ab56416; mouse monoclonal), and GAPDH (ab9485; rabbit polyclonal) were from Abcam; and galectin-3 (sc-32790; mouse monoclonal) and LC3B (L7543; rabbit polyclonal) were from Santa Cruz Biotechnology and MilliporeSigma, respectively.

Techniques: Plasmid Preparation, Injection, Western Blot, Control, Activity Assay, Staining