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

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: Molecular Medicine Reports

Article Title: ER-α36 knockdown is associated with lysosomal dysfunction and proliferation inhibition in liver cancer cells

doi: 10.3892/mmr.2025.13649

Figure Lengend Snippet: ER-α36 knockdown blocks autophagic flux and degradation. Expression of p62 in stably transfected liver cancer cells with different levels of ER-α36 expression: (A) Representative western blots and (B) quantitative analysis. (C) Confocal microscopy images of liver cancer cells with and without ER-α36 knockdown infected with pmCherry-enhanced GFP-LC3b adenovirus (scale bar, 10 µm) and (D) quantification of yellow and red puncta. (E) Levels of LC3-II and LC3-I assessed by western blotting in transfected liver cancer cells treated with or without CQ (20 µM) for 24 h, and (F) quantitative analysis of the LC3-II/LC3-I ratio. (G) Western blots of ubiquitinated proteins in the transfected liver cancer cells and (H) quantitative analysis of Ub levels. **P<0.01. ER, estrogen receptor; GFP, green fluorescence protein; LC3, microtubule-associated protein 1 light chain 3; CQ, chloroquine; Ub, ubiquitin; Sh36, transfected with ER-α36 specific short hairpin RNA expression vector; Vector, transfected with empty vector.

Article Snippet: Antibodies against p62 (also known as sequestosome 1; cat. no. 18420-1-AP), galectin-3 (Gal-3; cat. no. 60207-1-Ig), lysosome-associated membrane protein 1 (LAMP1; cat. no. 21997-1-AP), AKT (cat. no. 10176-2-AP), phosphorylated-(p-)AKT (cat. no. 66444-1-Ig) and β-actin (cat. no. 60008-1-IG) were purchased from Proteintech Group, Inc.

Techniques: Knockdown, Expressing, Stable Transfection, Transfection, Western Blot, Confocal Microscopy, Infection, Fluorescence, Ubiquitin Proteomics, shRNA, Plasmid Preparation

Journal: Molecular Medicine Reports

Article Title: ER-α36 knockdown is associated with lysosomal dysfunction and proliferation inhibition in liver cancer cells

doi: 10.3892/mmr.2025.13649

Figure Lengend Snippet: ER-α36 knockdown suppresses the malignant proliferation of liver cancer cells and induces lysosomal membrane permeabilization. (A) Representative images of liver tumors from nude mice intrahepatically injected with HepG2 cells expressing different ER-α36 levels, harvested at 28 days post-injection. (B) Quantitative analysis of liver tumor volume, liver weight, body weight and the ratio of liver weight to body weight. (C) Representative H&E staining images showing pathological karyomitosis changes. Scale bar, 50 µm. (D) Immunohistochemical staining of LC3, p62, LAMP1 and Ki67 with corresponding IOD values. Scale bar, 50 µm. (E) Immunofluorescence staining of Gal-3 in the tumors formed from the transfected HepG2 cells. Scale bar, 10 µm. (F) Western blotting results showing the levels of LC3-II and LC3-II, p62, LAMP1, p-AKT and t-AKT in the tumors, and (G) quantitative analyses of the LC3-II/LC3-I and p-AKT/t-AKT ratios, and p62 and LAMP1 expression levels. β-actin was used as the internal loading control. Data are presented as the mean ± SEM. **P<0.01. ER, estrogen receptor; H&E, hematoxylin and eosin; LC3, microtubule-associated protein 1 light chain 3; LAMP1, lysosome-associated membrane protein 1; Gal-3, galectin-3; IOD, integrated optical density; p-, phosphorylated; t-, total; Sh36, transfected with ER-α36 specific short hairpin RNA expression vector; Vector, transfected with empty vector.

Article Snippet: Antibodies against p62 (also known as sequestosome 1; cat. no. 18420-1-AP), galectin-3 (Gal-3; cat. no. 60207-1-Ig), lysosome-associated membrane protein 1 (LAMP1; cat. no. 21997-1-AP), AKT (cat. no. 10176-2-AP), phosphorylated-(p-)AKT (cat. no. 66444-1-Ig) and β-actin (cat. no. 60008-1-IG) were purchased from Proteintech Group, Inc.

Techniques: Knockdown, Membrane, Injection, Expressing, Staining, Immunohistochemical staining, Immunofluorescence, Transfection, Western Blot, Control, shRNA, Plasmid Preparation

Journal: Stem Cell Research & Therapy

Article Title: A serum-free adipose-conditioned medium delays stem cell senescence and maintains tissue homeostasis via IL-6/STAT3 axis suppression

doi: 10.1186/s13287-025-04721-8

Figure Lengend Snippet: SF-ACM is safe in vivo and improves aging-related skin and muscle structure. A and B Percentage change in body weight during SF-ACM intervention in the D-galactose–induced progeroid model and the naturally aged model (n = 6). P values were determined by two-way ANOVA. C – E Serum levels of BUN (mmol/L), ALT (IU/L), and AST (IU/L) in the progeroid model (n = 6). F – H Same analyses in the naturally aged model (n = 6). I Representative images of H&E and Masson’s trichrome staining of dorsal skin from both models. Scale bar, 200 μm. J and K Quantification of hair follicle density (n/mm 2 ) in the progeroid and naturally aged models (n = 4). L and M Quantification of epidermal thickness (μm) in the progeroid and naturally aged models (n = 5). N and O Quantification of dermal thickness (μm) in the progeroid and naturally aged models (n = 5). ( P ) Representative H&E images of gastrocnemius muscle cross-sections. Scale bar, 200 μm. Q and R Quantification of myofiber cross-sectional area (μm 2 ) in the progeroid and aturally aged models. n = 5. All data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Statistical significance was determined using an unpaired two-tailed t-test for the naturally aged models and one-way ANOVA for the progeroid models, unless otherwise specified. Y, young mice (4 months); A, aged mice (16 months); NS, normal saline; DGal, D-galactose

Article Snippet: For the D-galactose–induced aging model, 4-month-old mice were randomly assigned (using a random number table) to four groups (n = 6 per group) and treated for 8 weeks as follows: (1–2) Mice in the control groups received intraperitoneal injections of 100 μL per injection, once weekly, of either normal saline or low-glucose DMEM (C11885500BT; Gibco, Thermo Fisher Scientific, Waltham, MA, USA); (3–4) Mice in the D-galactose groups received the same weekly intraperitoneal injections (100 μL of low-glucose DMEM or SF-ACM), and additionally received daily subcutaneous injections of D-galactose (HY-N0210; MedChemExpress, Monmouth Junction, NJ, USA; 100 mg/kg/day, 100 μL per injection).

Techniques: In Vivo, Staining, Two Tailed Test, Saline

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: Journal of immunology (Baltimore, Md. : 1950)

Article Title: β -Catenin/Tcf Determines the Outcome of Thymic Selection in Response to αβ TCR Signaling 1

doi: 10.4049/jimmunol.0901369

Figure Lengend Snippet: TCR signals activate the β-catenin/Tcf pathway in DP cells. A, TopGal and wild-type thymocytes were stained with a fluorescent β-galactosidase substrate (FDG) followed by surface staining for CD4, CD8, and TCRβ. Gated DP, CD4, and CD8 subpopulations were analyzed for β-galactosidase activity vs TCR surface expression by FACS. x- and y-axis represent log10 fluorescence. B, Semiquantitative RT-PCR for Tcf-1 mRNA on 1/5 serial dilutions of target cDNA extracted from sorted DP (TCRlow) and (TCRhigh) cells. MHCI-II double-deficient mice were stimulated with anti-CD3 Abs in vivo by a single i.p. injection (50 μg/mouse) or DP thymocytes from MHCI-II double-deficient mice were sorted for ex vivo stimulation either with plate-bound (coated) or soluble anti-CD3 (1 g/ml) plus anti-CD28 Abs (5 μg/ml). C, The kinetics of activation were followed by surface expression of CD69 and TCR at the indicated times and treatment by FACS (log10 fluorescence). D, Representative immunoblot for β-catenin and GADPH protein levels after in vivo or ex vivo stimulation of DP cells as for the indicated times. E, Densitometric quantification of β-catenin vs GADPH levels from three to five immunoblots as in D using the ImageJ software (average ± SE). t test in vivo 0 vs 2 or 6 h, p = 0.003 and p = 0.007, respectively; ex vivo (coated) 0 vs 2 or 4 h, p = 0.009 and p = 0.04, respectively.*, Statistically significant induction (p<0.05). F, Sorted DP thymocytes were stimulated as indicated for 2 h before processing for Western blot analysis. Histograms represent densitometric quantification of β-catenin levels relative to GAPDH from three independent immunoblots using the ImageJ software (average ± SD).*, A statistically significant induction (t test) compared with nonstimulated cells; p<0.05. The gel below the histograms shows a representative experiment. G, Jurkat cells and derivative cell lines were induced by plate-bound anti-CD3 plus anti-CD28 Abs as indicated. Whole-cell lysates were prepared at the indicated time points after TCR stimulation and used in Western blot analyses to detect β-catenin or GAPDH. Densitometric quantification of β-catenin levels relative to GAPDH of three to five independent blots (average ± SD).*, A statistically significant induction (t test) compared with nonstimulated cells at 1 h, p = 0.05, and at 3 h, p = 0.01.

Article Snippet: Briefly, 10 6 thymocytes suspended in FACS buffer were subjected to a hypotonic shock for 40 s at 37°C by adding an equal volume of a solution containing the fluorescent substrate fluorescein-di- β -D-galactopyranoside (FDG) (Invitrogen) in distilled water.

Techniques: Staining, Activity Assay, Expressing, Fluorescence, Reverse Transcription Polymerase Chain Reaction, In Vivo, Injection, Ex Vivo, Activation Assay, Western Blot, Software

Journal: Clinical and Translational Medicine

Article Title: P2X7R deficiency alleviates cardiac senescence by enhancing mitophagy via the HuR/TRIM26/NR4A1 axis

doi: 10.1002/ctm2.70621

Figure Lengend Snippet: Purinergic 2×7 receptor (P2X7R) expression in the serum of elderly humans and heart tissues of aged mice. (A) P2X7R levels in the serum of humans in three age groups: young, middle‐aged and elderly ( n = 40). (B) Correlation between P2X7R serum expression and human age. (C) Transcriptome sequencing data showing differential P2X7R gene expression in the heart tissues of young and old mice. (D) Representative images of P2X7R immunoreactivity in the myocardia of 3‐, 10‐ and 24‐month‐old mice (scale bar, 50 µm) ( n = 6). (E) Semiquantitative analysis of the P2X7R area ratio in (D) ( n = 6). (F) P2X7R expression in the myocardial tissue of the wild type (WT)‐young and WT‐old groups. GAPDH was used as a loading control ( n = 6). (G) Densitometric quantification of the immunoblots in (F) ( n = 6). (H) mRNA levels of P2X7R in the myocardial tissue of the WT‐young and WT‐old groups ( n = 6). (I) Representative Western blot analysis of P2X7R levels in HL‐1 cells treated with 0, 5, 10 or 15 g/L D‐galactose (D‐gal). GAPDH was used as a loading control ( n = 3). (J) Densitometric quantification of the immunoblots in (I) ( n = 3). (K) Representative Western blot analysis of tumour protein 53 (P53), CDN1A (P21) and p16INK4a (P16) levels in D‐gal‐induced HL‐1 cells pretreated with A438079 or DMSO. GAPDH was used as a loading control ( n = 3). (L) Representative senescence‐associated β‐galactosidase (SA‐β‐Gal) staining images of HL‐1 cells ( n = 3). (M) Percentages of SA‐β‐Gal+ cells in (L) were quantified ( n = 3). Adjusted p ‐values are provided in the case of multiple group comparisons. P2X7R, purinergic receptor P2X, ligand‐gated ion channel 7; young, 3 months old; old, 24 months old; A438079, P2X7R inhibitor. DMSO, Dimethyl Sulfoxide; GAPDH, Glyceraldehyde‐3‐Phosphate Dehydrogenase.

Article Snippet: Two weeks later, the mice received daily subcutaneous injections of D‐galactose (D‐gal, 300 mg/kg/day; Sangon Biotech) for 6 weeks to establish a D‐gal‐induced ageing model. Control animals were administered an equivalent volume of saline.

Techniques: Expressing, Sequencing, Gene Expression, Control, Western Blot, Staining

Journal: Clinical and Translational Medicine

Article Title: P2X7R deficiency alleviates cardiac senescence by enhancing mitophagy via the HuR/TRIM26/NR4A1 axis

doi: 10.1002/ctm2.70621

Figure Lengend Snippet: Purinergic 2×7 receptor (P2X7R) deficiency increases nuclear receptor subfamily 4 group A member 1 (NR4A1) expression to alleviate ageing‐induced cardiac mitophagy. (A) Heatmap showing differentially expressed genes in wild type (WT)‐old and P2X7R −/− ‐old mouse hearts ( n = 3). (B and C) Representative Western blot analysis of NR4A1 expression in the myocardial tissue of WT‐young, WT‐old and P2X7R −/− ‐old mice; GAPDH was used as a loading control (B) and density analysis (C) ( n = 6). (D) NR4A1 expression in the Ctrl, A438079, D‐galactose (D‐gal) and A438079 + D‐gal groups and density analysis ( n = 3). (E and F) Representative Western blot analysis of tumour protein 53 (P53), CDN1A (P21) and p16INK4a (P16) levels in D‐gal‐induced HL‐1 cells treated with P2X7R overexpression (P2X7R oe ) or NR4A1 overexpression (NR4A1 oe ); GAPDH was used as a loading control (E) and densitometric quantification (F) ( n = 3). (G and H) Representative Western blot analysis of sequestosome‐1, SQSTM1 (P62), PTEN‐induced putative kinase 1 (PINK1), PARK2 (Parkin) and LC3B (microtubule‐associated protein 1 light chain 3B) levels in D‐gal‐induced HL‐1 cells treated with P2X7R oe or NR4A1 oe ( n = 3). GAPDH was used as a loading control (G) and density analysis (H). GAPDH was used as a loading control. (I) Representative images of SA‐β‐Gal staining of HL‐1 cells ( n = 3). (J) The percentages of SA‐β‐Gal+ cells in (I) were quantified ( n = 3). (K) Representative confocal images of mt‐Keima expression in each group ( n = 3). (L) Double immunofluorescence staining for Parkin (red) and TOMM20 (green) in HL‐1 cells treated with D‐gal, NR4A1 oe or P2X7R oe ( n = 3). Merged images (orange) show colocalisation (scale bar, 20 µm). Adjusted p ‐values are provided in the case of multiple group comparisons. P2X7R −/− , mice with whole‐body P2X7R knockout. GAPDH, Glyceraldehyde‐3‐Phosphate Dehydrogenase.

Article Snippet: Two weeks later, the mice received daily subcutaneous injections of D‐galactose (D‐gal, 300 mg/kg/day; Sangon Biotech) for 6 weeks to establish a D‐gal‐induced ageing model. Control animals were administered an equivalent volume of saline.

Techniques: Expressing, Western Blot, Control, Over Expression, Staining, Double Immunofluorescence Staining, Knock-Out

Journal: Clinical and Translational Medicine

Article Title: P2X7R deficiency alleviates cardiac senescence by enhancing mitophagy via the HuR/TRIM26/NR4A1 axis

doi: 10.1002/ctm2.70621

Figure Lengend Snippet: Overexpression of nuclear receptor subfamily 4 group A member 1 (NR4A1) eliminates Purinergic 2×7 receptor (P2X7R)‐mediated cardiac remodelling and mitophagy in D‐galactose (D‐gal)‐induced mice. (A) Schematic diagram depicting the experimental strategy for the D‐gal‐induced ageing model and the activation of NR4A1 and P2X7R. (B) Representative M‐mode echocardiographic images of the left ventricle from D‐gal + EV, D‐gal + P2X7R oe , D‐gal + NR4A1 oe or D‐gal + P2X7R oe + NR4A1 oe mice ( n = 8). (C and D) LV ejection fraction and fractional shortening were assessed by echocardiography ( n = 8). (E and F) Left ventricular anterior wall thickness (left ventricular anterior wall in systole [LVAWs] and left ventricular anterior wall in diastole [LVAWd]) was assessed by echocardiography ( n = 8). (G) Representative images of haematoxylin and eosin (H&E) staining (scale bar, 50 µm). (H and I) Representative images of Masson staining (H) (scale bar, 50 µm) and quantification of the interstitial fibrotic area (I) ( n = 6). (J and K) Representative images of Sirius Red staining (J) (scale bar, 50 µm) and quantification of the interstitial fibrotic area (K) ( n = 6). (L and M) Representative images of wheat germ agglutinin (WGA)‐stained sections (L) (scale bar, 50 µm) and quantification of cardiomyocyte cross‐sections (M) ( n = 6). (N and O) Representative images of β‐Gal immunoreactivity in myocardial tissue (scale bar, 50 µm) and quantification of the percentage of the β‐Gal + area (O) ( n = 6). (P) Representative Western blot analysis of tumour protein 53 (P53), CDN1A (P21) and p16INK4a (P16) levels in the myocardial tissue of D‐gal + EV, D‐gal + P2X7R oe , D‐gal + NR4A1 oe and D‐gal + P2X7R oe + NR4A1 oe mice. GAPDH was used as a loading control ( n = 6). (Q) Representative Western blot analysis of sequestosome‐1, SQSTM1 (P62), PTEN‐induced putative kinase 1 (PINK1), PARK2 (Parkin) and LC3B (microtubule‐associated protein 1 light chain 3B) levels in the myocardial tissue of D‐gal + EV, D‐gal + P2X7R oe , D‐gal + NR4A1 oe and D‐gal + P2X7R oe + NR4A1 oe mice. GAPDH was used as a loading control ( n = 6). Adjusted p ‐values are provided in the case of multiple group comparisons. P2X7R oe , AAV9‐cTnT‐P2X7R; NR4A1 oe , AAV9‐cTnT‐NR4A1; EV, AAV9‐cTnT‐EV. GAPDH, Glyceraldehyde‐3‐Phosphate Dehydrogenase.

Article Snippet: Two weeks later, the mice received daily subcutaneous injections of D‐galactose (D‐gal, 300 mg/kg/day; Sangon Biotech) for 6 weeks to establish a D‐gal‐induced ageing model. Control animals were administered an equivalent volume of saline.

Techniques: Over Expression, Activation Assay, Staining, Western Blot, Control

Journal: Clinical and Translational Medicine

Article Title: P2X7R deficiency alleviates cardiac senescence by enhancing mitophagy via the HuR/TRIM26/NR4A1 axis

doi: 10.1002/ctm2.70621

Figure Lengend Snippet: Tripartite motif containing 26 (TRIM26) directly binds to nuclear receptor subfamily 4 group A member 1 (NR4A1) and promotes its degradation. (A) Heatmap of TRIM family expression profiles in mouse models following ageing from the GSE175854 dataset ( n = 2). (B) mRNA levels of TRIM26, TRIM55, TRIM71, TRIM41, TRIM16, TRIM30a, TRIM34a, TRIM65, TRIM2 and TRIM5 in the myocardial tissue of wild type (WT)‐young, WT‐old and P2X7R −/− ‐old mice ( n = 6). (C) TRIM26 expression in the myocardial tissue of WT‐young, WT‐old and P2X7R −/− ‐old mice; GAPDH was used as a loading control; and density analysis ( n = 6). (D) Double immunofluorescence staining for TRIM26 (red) and NR4A1 (green) in HL‐1 cells treated with D‐galactose (D‐gal) or A438079. Merged images (orange) show colocalisation (scale bar, 10 µm). (E and F) Coimmunoprecipitation (Co‐IP) experiments of TRIM26 and NR4A1 in myocardial tissue (E) and HL‐1 cells (F). (G) Protein level of NR4A1 in HL‐1 cells after treatment with cycloheximide (CHX) and density analysis ( n = 3). (H) Ubiquitination level of NR4A1 under TRIM26 silencing (TRIM26 si ) plasmid treatment. Adjusted p ‐values are provided in the case of multiple group comparisons. P2X7R −/− , mice with whole‐body P2X7R knockout; TRIM26 oe , TRIM26 overexpression. GAPDH, Glyceraldehyde‐3‐Phosphate Dehydrogenase.

Article Snippet: Two weeks later, the mice received daily subcutaneous injections of D‐galactose (D‐gal, 300 mg/kg/day; Sangon Biotech) for 6 weeks to establish a D‐gal‐induced ageing model. Control animals were administered an equivalent volume of saline.

Techniques: Expressing, Control, Double Immunofluorescence Staining, Co-Immunoprecipitation Assay, Ubiquitin Proteomics, Plasmid Preparation, Knock-Out, Over Expression

Journal: Clinical and Translational Medicine

Article Title: P2X7R deficiency alleviates cardiac senescence by enhancing mitophagy via the HuR/TRIM26/NR4A1 axis

doi: 10.1002/ctm2.70621

Figure Lengend Snippet: Purinergic 2×7 receptor (P2X7R) decreased tripartite motif containing 26 (TRIM26) mRNA degradation in ageing hearts by affecting human antigen R (HuR). (A) D‐galactose (D‐gal)‐induced HL‐1 cells were pretreated with DMSO or A438079, treated with actinomycin D and monitored for 2, 4, 6 and 8 h, after which the mRNA expression of TRIM26 was measured by RT‒qPCR ( n = 3). (B) Schematic representation of the TRIM26 mRNA structure. (C) HuR expression in the nuclei of HL‐1 cells treated with D‐gal or A438079. Histone H3 was used as a loading control for the nuclear expression of HuR, and GAPDH was used as a loading control for the cytoplasmic and total expression of HuR ( n = 3). (D–F) Densitometric quantification of the immunoblots in (C). (G) Immunofluorescence staining for HuR (green) in HL‐1 cells treated with D‐gal or A438079 (scale bar, 50 µm). (H and I) Protein and mRNA expression of HuR in the myocardial tissue of wild type (WT)‐young, WT‐old and P2X7R −/− ‐old mice and density analysis ( n = 6). (J) Myocardial tissue of WT‐old, WT‐old and P2X7R −/− ‐old mice was subjected to RNA immunoprecipitation for TRIM26 mRNA using an anti‐HuR antibody ( n = 6). Adjusted p ‐values are provided in the case of multiple group comparisons. P2X7R −/− , mice with whole‐body P2X7R knockout. DMSO, Dimethyl Sulfoxide; GAPDH, Glyceraldehyde‐3‐Phosphate Dehydrogenase.

Article Snippet: Two weeks later, the mice received daily subcutaneous injections of D‐galactose (D‐gal, 300 mg/kg/day; Sangon Biotech) for 6 weeks to establish a D‐gal‐induced ageing model. Control animals were administered an equivalent volume of saline.

Techniques: Expressing, Control, Western Blot, Immunofluorescence, Staining, RNA Immunoprecipitation, Knock-Out

Journal: PLoS ONE

Article Title: Expression and Functions of Galectin-7 in Human and Murine Melanomas

doi: 10.1371/journal.pone.0063307

Figure Lengend Snippet: A) Percentage of positive biopsies of normal skin (n = 7), nevus (n = 18) and malignant melanoma (n = 45), as determined by the in silico analysis of galectin-7 expression from a microarray of human biopsies . B) Representative graph of galectin-7 mRNA expression in each biopsy described in (A). C) Detection of galectin-7 by immunohistochemistry in representative biopsies of nevi and malignant melanoma. Overall, 13 malignant melanomas and 47 nevi were tested.

Article Snippet: Anti-cleaved PARP-1 antibody was purchased from Epitomics (Burlingame, CA); anti-β-actin antibody was purchased from Sigma-Aldrich (St. Louis, MO); anti-EGR-1 antibody was purchased from Santa Cruz (CA, USA); anti-human galectin-7 monoclonal antibody was purchased from R & D Systems; methyl [ 3 H]thymidine was purchased from Perkin Elmer (Waltham, MA); cell culture lysis reagent (CCLR) and passive lysis buffer were purchased from Promega (Madison, WI); RIPA lysis buffer was purchased from Thermo Scientific (Rockford, USA); and buffered formaldehyde solution was purchased from Fisher Scientific (Toronto, ON).

Techniques: In Silico, Expressing, Microarray, Immunohistochemistry

Journal: PLoS ONE

Article Title: Expression and Functions of Galectin-7 in Human and Murine Melanomas

doi: 10.1371/journal.pone.0063307

Figure Lengend Snippet: Primary tumors were collected at necropsy 18 days after the subcutaneous injection of B16F1 cells (5×10 4 cells) in C57BL/6 (WT) and galectin-7-deficient mice (KOG7). (A) RT-PCR analysis of galectin-7 mRNA expression in two B16F1 primary tumors from WT and KOG7 mice in comparison with the B16F1 cell line. Actin was used as a loading and specificity control. B) Immunohistochemistry for galectin-7 in normal skin (i, ii) and B16F1 primary tumors (iii, iv) in WT and KOG7 mice. These galectin-7-positive structures located in the suprabasal epidermis have been reported before and likely represent suprabasal keratinocytes, which are known to express galectin-7 constitutively . Control stained without HRP and in absence of Abs did not show any detectable staining in both wt and KOG7 mice. C) Immunohistochemistry for galectin-7 in a lung collected 20 days post-injection from one KOG7 mouse injected intravenously via the tail vein with B16F1 cells (2×10 5 cells) in comparison with a normal lung. Scale bars in all immunohistochemistry images represent 600 µM.

Article Snippet: Anti-cleaved PARP-1 antibody was purchased from Epitomics (Burlingame, CA); anti-β-actin antibody was purchased from Sigma-Aldrich (St. Louis, MO); anti-EGR-1 antibody was purchased from Santa Cruz (CA, USA); anti-human galectin-7 monoclonal antibody was purchased from R & D Systems; methyl [ 3 H]thymidine was purchased from Perkin Elmer (Waltham, MA); cell culture lysis reagent (CCLR) and passive lysis buffer were purchased from Promega (Madison, WI); RIPA lysis buffer was purchased from Thermo Scientific (Rockford, USA); and buffered formaldehyde solution was purchased from Fisher Scientific (Toronto, ON).

Techniques: Injection, Reverse Transcription Polymerase Chain Reaction, Expressing, Comparison, Immunohistochemistry, Staining

Journal: PLoS ONE

Article Title: Expression and Functions of Galectin-7 in Human and Murine Melanomas

doi: 10.1371/journal.pone.0063307

Figure Lengend Snippet: A) RT-PCR analysis for galectin-7 mRNA expression in transfectant cells overexpressing galectin-7 with or without luciferase in comparison with a control B16F1 cell line (F1). The aggressive murine lymphoma cell line S19 (+) was used as a positive control. Actin was used as a loading and specificity control. B) Luciferase assay of three B16F1 transfectant cell lines overexpressing luciferase and cotransfected with pRc-CMV2-galectin-7 in comparison with the control B16F1 cell line. C) Confocal microscopy for galectin-7 in control B16F1 cells (iii) and galectin-7 transfectant cells (G7#5) (iv); these cells were also visualized (i, ii).

Article Snippet: Anti-cleaved PARP-1 antibody was purchased from Epitomics (Burlingame, CA); anti-β-actin antibody was purchased from Sigma-Aldrich (St. Louis, MO); anti-EGR-1 antibody was purchased from Santa Cruz (CA, USA); anti-human galectin-7 monoclonal antibody was purchased from R & D Systems; methyl [ 3 H]thymidine was purchased from Perkin Elmer (Waltham, MA); cell culture lysis reagent (CCLR) and passive lysis buffer were purchased from Promega (Madison, WI); RIPA lysis buffer was purchased from Thermo Scientific (Rockford, USA); and buffered formaldehyde solution was purchased from Fisher Scientific (Toronto, ON).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Transfection, Luciferase, Comparison, Positive Control, Confocal Microscopy

Journal: PLoS ONE

Article Title: Expression and Functions of Galectin-7 in Human and Murine Melanomas

doi: 10.1371/journal.pone.0063307

Figure Lengend Snippet: A) Images of the motility assay after the wounding of confluent B16F1 cells overexpressing galectin-7 (B16-G7) or controls (B16-Srα) at T = 0 and T = 16 hr. A mixture of three clones of B16F1-G7 was used (G7 #5, 10 and 14). B) Representative graph of the results obtained in (A).

Article Snippet: Anti-cleaved PARP-1 antibody was purchased from Epitomics (Burlingame, CA); anti-β-actin antibody was purchased from Sigma-Aldrich (St. Louis, MO); anti-EGR-1 antibody was purchased from Santa Cruz (CA, USA); anti-human galectin-7 monoclonal antibody was purchased from R & D Systems; methyl [ 3 H]thymidine was purchased from Perkin Elmer (Waltham, MA); cell culture lysis reagent (CCLR) and passive lysis buffer were purchased from Promega (Madison, WI); RIPA lysis buffer was purchased from Thermo Scientific (Rockford, USA); and buffered formaldehyde solution was purchased from Fisher Scientific (Toronto, ON).

Techniques: Motility Assay, Clone Assay

Journal: PLoS ONE

Article Title: Expression and Functions of Galectin-7 in Human and Murine Melanomas

doi: 10.1371/journal.pone.0063307

Figure Lengend Snippet: B16F1 cells overexpressing galectin-7 (+) or controls (–) were treated with various doses of quercetin. A) Apoptotic sensitivity was analyzed by western blotting for cleaved PARP-1 detection. B) RT-PCR analysis of galectin-7 and EGR-1 mRNA expression. C) Western blot analysis for EGR-1 detection, 293 cells transfected with EGF were used as a positive control. Actin was used as a loading and specificity control. D) Dual luciferase assay of B16F1 transfectant cells overexpressing galectin-7 (□) or controls (▪) co-transfected with luciferase reporter plasmids with an EGR-1 promoter and pRLSV40-Renilla vector as a transfection control and treated with various doses of quercetin for 24 h. A mixture of three clones of B16F1-G7 was used (G7 #5, 10 and 14) for all of these experiments.

Article Snippet: Anti-cleaved PARP-1 antibody was purchased from Epitomics (Burlingame, CA); anti-β-actin antibody was purchased from Sigma-Aldrich (St. Louis, MO); anti-EGR-1 antibody was purchased from Santa Cruz (CA, USA); anti-human galectin-7 monoclonal antibody was purchased from R & D Systems; methyl [ 3 H]thymidine was purchased from Perkin Elmer (Waltham, MA); cell culture lysis reagent (CCLR) and passive lysis buffer were purchased from Promega (Madison, WI); RIPA lysis buffer was purchased from Thermo Scientific (Rockford, USA); and buffered formaldehyde solution was purchased from Fisher Scientific (Toronto, ON).

Techniques: Western Blot, Reverse Transcription Polymerase Chain Reaction, Expressing, Transfection, Positive Control, Luciferase, Plasmid Preparation, Clone Assay

Journal: PLoS ONE

Article Title: Expression and Functions of Galectin-7 in Human and Murine Melanomas

doi: 10.1371/journal.pone.0063307

Figure Lengend Snippet: A) Survival curve of C57BL/6 mice injected i.v. with B16F1 transfectant cells overexpressing galectin-7 (□) or controls (♦) (2×10 5 cells) (n = 8−10). B) Luciferase assay of lungs of C57BL/6 mice sacrificed at 9, 12, 15 or 18 days after the i.v. injection of a mixture of B16F1 luciferase transfectant cells overexpressing galectin-7 (+) or control (–) (n = 3−8). Normal lungs were used as a negative control (T: 0; n = 2). C) Ex vivo imaging of a lung metastasis, as seen in (B), of B16F1 cells overexpressing galectin-7 (iii and v) or control cells (ii and iv) at 9 and 18 days in comparison with control lungs (i).

Article Snippet: Anti-cleaved PARP-1 antibody was purchased from Epitomics (Burlingame, CA); anti-β-actin antibody was purchased from Sigma-Aldrich (St. Louis, MO); anti-EGR-1 antibody was purchased from Santa Cruz (CA, USA); anti-human galectin-7 monoclonal antibody was purchased from R & D Systems; methyl [ 3 H]thymidine was purchased from Perkin Elmer (Waltham, MA); cell culture lysis reagent (CCLR) and passive lysis buffer were purchased from Promega (Madison, WI); RIPA lysis buffer was purchased from Thermo Scientific (Rockford, USA); and buffered formaldehyde solution was purchased from Fisher Scientific (Toronto, ON).

Techniques: Injection, Transfection, Luciferase, Negative Control, Ex Vivo, Imaging, Comparison

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