Journal: Archives of Gynecology and Obstetrics

Article Title: Glycosylated fibronectin as a first trimester marker for gestational diabetes

doi: 10.1007/s00404-020-05670-8

Figure Lengend Snippet: a Glycosylated fibronectin concentrations in BMI subgroups of 20–25 and 30–35 kg/m 2 in the study and the control group. b Effect of smoking on concentrations of glycosylated fibronectin in the control and GDM groups. c Multiple of median (MoM) values for fibronectin and glycosylated fibronectin between control and GDM groups. d Multiple of median (MoM) values for adiponectin and glycosylated adiponectin between control and GDM groups

Article Snippet: Calibrators for both assays were made from recombinant human adiponectin (1065-AF, R&D Systems, Abingdon, UK).

Techniques: Control

Journal: Journal of Inflammation Research

Article Title: Unleashing AdipoRon’s Potential: A Fresh Approach to Tackle Pseudomonas aeruginosa Infections in Bronchiectasis via Sphingosine Metabolism Modulation

doi: 10.2147/JIR.S483689

Figure Lengend Snippet: The expression of adiponectin and AdipoR1 within bronchial epithelium determined in Pseudomonas aeruginosa infected mouse models. ( A ) Comparison of the expression of adiponectin between Pseudomonas aeruginosa infected mouse models and the uninfected mouse models by immunofluorescence (×200). ( B ) Comparison of the expression of AdipoR1 between Pseudomonas aeruginosa infected mouse models and the uninfected mouse models by immunofluorescence (×200). ( C ) Comparison of the expression of AdipoR1 between Pseudomonas aeruginosa infected mouse models and the uninfected mouse models by Western blot. ( D ) The expression levels of AdipoR1 in the bronchial epithelium of mice in different groups (DMSO control group, AdipoRon high-dose group, AdipoRon low-dose group and adiponectin Acrp30 recombinant protein group) were compared by immunofluorescence (×200). Data are expressed as the means ± standard deviation of three independent experiments. **indicates P <0.01.

Article Snippet: Treatment of the five groups: (i) Uninfected control: mice were anesthetized by intraperitoneal injection and instilled with 35 μL of PBS via the airway; (ii) Pseudomonas aeruginosa infection with DMSO control: 8 hours prior, mice were given an intravenous injection of DMSO solution as a control; (iii) Pseudomonas aeruginosa infection with low-dose AdipoRon: 8 hours prior, mice were treated with AdipoRon (5 mg/kg, intravenous injection); (iv) Pseudomonas aeruginosa infection with high-dose AdipoRon: 8 hours prior, mice were treated with AdipoRon (50 mg/kg, intravenous injection); and (v) Pseudomonas aeruginosa infection with adiponectin Acrp30 recombinant protein: 4 hours prior, mice were treated with adiponectin Acrp30 recombinant protein (HY-P7358, MedChemExpress, USA, 1 mg/kg, intravenous injection) as described.

Techniques: Expressing, Infection, Comparison, Immunofluorescence, Western Blot, Control, Recombinant, Standard Deviation

Journal: Journal of Inflammation Research

Article Title: Unleashing AdipoRon’s Potential: A Fresh Approach to Tackle Pseudomonas aeruginosa Infections in Bronchiectasis via Sphingosine Metabolism Modulation

doi: 10.2147/JIR.S483689

Figure Lengend Snippet: AdipoRon reduced the load of Pseudomonas aeruginosa in the airway of mice. ( A ) HE staining was performed on lung tissue specimens from mouse models with different treatment (control group without Pseudomonas aeruginosa infection, DMSO control group with Pseudomonas aeruginosa infection, AdipoRon low-dose (5 mg/kg) pretreatment group with Pseudomonas aeruginosa infection, AdipoRon high-dose (50 mg/kg) pretreatment group with Pseudomonas aeruginosa infection, and ACRP30 recombinant protein pretreatment group with Pseudomonas aeruginosa infection. ( B ) A semi-quantitative grading method of inflammation score was used to evaluate the severity of peribronchial inflammatory cell infiltration: 0, normal; 1, few cells; 2, a ring of inflammatory cells 1 cell layer deep; 3, a ring of inflammatory cells 2–4 cells deep; 4, a ring of inflammatory cells of > 4 cells deep. The inflammation scores of different groups were compared. ( C and D ) The CFU of Pseudomonas aeruginosa was counted in the lung tissue homogenates of mice in different treatment groups. ( E ) The bacterial load of Pseudomonas aeruginosa in the lung tissue of mice in different treatment groups was detected by immunofluorescence with the antibody against Pseudomonas aeruginosa (PA1-73116). Data are expressed as the means ± standard deviation of three independent experiments. **indicates P <0.01.

Article Snippet: Treatment of the five groups: (i) Uninfected control: mice were anesthetized by intraperitoneal injection and instilled with 35 μL of PBS via the airway; (ii) Pseudomonas aeruginosa infection with DMSO control: 8 hours prior, mice were given an intravenous injection of DMSO solution as a control; (iii) Pseudomonas aeruginosa infection with low-dose AdipoRon: 8 hours prior, mice were treated with AdipoRon (5 mg/kg, intravenous injection); (iv) Pseudomonas aeruginosa infection with high-dose AdipoRon: 8 hours prior, mice were treated with AdipoRon (50 mg/kg, intravenous injection); and (v) Pseudomonas aeruginosa infection with adiponectin Acrp30 recombinant protein: 4 hours prior, mice were treated with adiponectin Acrp30 recombinant protein (HY-P7358, MedChemExpress, USA, 1 mg/kg, intravenous injection) as described.

Techniques: Staining, Control, Infection, Recombinant, Immunofluorescence, Standard Deviation

Journal: Journal of Inflammation Research

Article Title: Unleashing AdipoRon’s Potential: A Fresh Approach to Tackle Pseudomonas aeruginosa Infections in Bronchiectasis via Sphingosine Metabolism Modulation

doi: 10.2147/JIR.S483689

Figure Lengend Snippet: AdipoRon increased sphingosine level in the lung of mouse infection model. ( A )The levels of sphingosine were compared between the Pseudomonas aeruginosa -infected mouse model group and the uninfected control group. ( B ) The metabolites in the lung tissue of mice in 3 groups were compared by PCA analysis: DMSO control group, adipoRon low-dose group and adipoRon high-dose group. ( C ) The levels of sphingosine in 3 groups were compared. ( D ) The metabolites in the lung tissue of mice were compared between adiponectin Acrp30 recombinant protein pretreatment group and the DMSO control group by PCA analysis. ( E ) The levels of sphingosine in 2 groups were compared. *** P <0.001.

Article Snippet: Treatment of the five groups: (i) Uninfected control: mice were anesthetized by intraperitoneal injection and instilled with 35 μL of PBS via the airway; (ii) Pseudomonas aeruginosa infection with DMSO control: 8 hours prior, mice were given an intravenous injection of DMSO solution as a control; (iii) Pseudomonas aeruginosa infection with low-dose AdipoRon: 8 hours prior, mice were treated with AdipoRon (5 mg/kg, intravenous injection); (iv) Pseudomonas aeruginosa infection with high-dose AdipoRon: 8 hours prior, mice were treated with AdipoRon (50 mg/kg, intravenous injection); and (v) Pseudomonas aeruginosa infection with adiponectin Acrp30 recombinant protein: 4 hours prior, mice were treated with adiponectin Acrp30 recombinant protein (HY-P7358, MedChemExpress, USA, 1 mg/kg, intravenous injection) as described.

Techniques: Infection, Control, Recombinant

Journal: Journal of Inflammation Research

Article Title: Unleashing AdipoRon’s Potential: A Fresh Approach to Tackle Pseudomonas aeruginosa Infections in Bronchiectasis via Sphingosine Metabolism Modulation

doi: 10.2147/JIR.S483689

Figure Lengend Snippet: AdipoRon activated P-AMPKα/PGC1α, inhibited TLR4/P-NF-κB p65, and reduced expression of bax in Pseudomonas aeruginosa -infected mouse model. ( A and B ) Western blot was used to quantitatively analyze the expression levels of several major indicators in the lung tissues of mice in three treatment groups (DMSO control group, AdipoRon low-dose group and AdipoRon high-dose group). Image J software was used to analyze the gray levels of different protein imprinting bands and to make statistical analysis. ( C and D ) Western blot was used to quantitatively analyze the expression levels of several major indexes in the lung tissues of mice in two groups: DMSO control group and Acrp30 recombinant protein pretreatment group. Data are expressed as the means ± standard deviation of three independent experiments. *indicates P <0.05, **indicates P <0.01.

Article Snippet: Treatment of the five groups: (i) Uninfected control: mice were anesthetized by intraperitoneal injection and instilled with 35 μL of PBS via the airway; (ii) Pseudomonas aeruginosa infection with DMSO control: 8 hours prior, mice were given an intravenous injection of DMSO solution as a control; (iii) Pseudomonas aeruginosa infection with low-dose AdipoRon: 8 hours prior, mice were treated with AdipoRon (5 mg/kg, intravenous injection); (iv) Pseudomonas aeruginosa infection with high-dose AdipoRon: 8 hours prior, mice were treated with AdipoRon (50 mg/kg, intravenous injection); and (v) Pseudomonas aeruginosa infection with adiponectin Acrp30 recombinant protein: 4 hours prior, mice were treated with adiponectin Acrp30 recombinant protein (HY-P7358, MedChemExpress, USA, 1 mg/kg, intravenous injection) as described.

Techniques: Expressing, Infection, Western Blot, Control, Software, Recombinant, Standard Deviation

Journal: PLoS ONE

Article Title: Globular Adiponectin Causes Tolerance to LPS-Induced TNF-α Expression via Autophagy Induction in RAW 264.7 Macrophages: Involvement of SIRT1/FoxO3A Axis

doi: 10.1371/journal.pone.0124636

Figure Lengend Snippet: (A and B) RAW 264.7 macrophages were treated with gAcrp (1 μg/ml) for different time duration (A) or different concentrations for 24 h (B). LC3II expression level was determined by Western blot analysis as described in materials and methods. (C and D) Cells were treated with gAcrp (1 μg/ml) for the indicated time periods (C) or different concentrations for 24 h (D). Atg5 expression level was examined by Western blot analysis as described previously. (E) Cells were treated with gAcrp (1 μg/ml) for the indicated time periods. Beclin-1 expression level was determined by Western blot analysis. Representative image from three independent experiments has been shown along with β-actin as internal loading control. (F) Cells were pretreated with Bafilomycin A1 (10 nM) for 2 h, followed by treatment with gAcrp (1 μg/ml) for additional 24 h. LC3II protein level was examined by Western blot analysis as described previously. Images are representative of three independent experiments that showed similar results. (G) Cells were transiently transfected with eGFP-LC3 plasmid. After 48 h, cells were treated with indicated concentration of gAcrp for 24 h. GFP-LC3 dots formation was viewed with A1 Confocal Laser Microscope System as described in material and methods. Representative image from three independent experiments has been shown along with quantitation of LC3 dots (lower panel). Values are expressed as percentage of cells with GFP-LC3 dots obtained from at least 100 cells. (H, I and J) Primary peritoneal macrophages were isolated from mice as indicated in materials and methods. Treatment was done identical to those outlined in Fig 1A and B. Expression levels of LC3II (H), Atg5 (I) and Beclin-1 (J) were measured by Western blot analysis. Representative images are shown along with β-actin as internal loading control. Quantitative analysis of LC3II and Atg5 expression was performed by densitometric analysis and is shown in bar graph (lower panel). Values are presented as mean ± SEM. * P < 0.05 compared to control.

Article Snippet: Recombinant human globular adiponectin (gAcrp) was acquired from Peprotech Inc. (Rocky Hill, NJ, USA) and 5-Chloromethyl-2, 7-dichlorodihydrofluorescein diacetate (CMH2DCFDA) from Molecular Probes (Eugene, OR).

Techniques: Expressing, Western Blot, Control, Transfection, Plasmid Preparation, Concentration Assay, Microscopy, Quantitation Assay, Isolation

Journal: PLoS ONE

Article Title: Globular Adiponectin Causes Tolerance to LPS-Induced TNF-α Expression via Autophagy Induction in RAW 264.7 Macrophages: Involvement of SIRT1/FoxO3A Axis

doi: 10.1371/journal.pone.0124636

Figure Lengend Snippet: Conditioned medium (CM) were prepared as described in materials and methods. After treatment with CM for 24 h, LC3II (A) and Atg5 (B) protein expression levels were determined by Western blot analysis. Images are representative of three independent experiments. Quantitative analyses of LC3II (A) and Atg5 (B) protein expression were performed by densitometric analysis and are shown in the graph (lower panel). Values are presented as mean ± SEM (n = 3). * P < 0.05 compared to control and CM-U. Control; Cells treated with DMEM containing 0.1% FBS, CM-U; CM prepared from gAcrp unstimulated cells, CM-12; Cells were stimulated with gAcrp for 8 h. After changing medium, cells were further incubated with 0.1% FBS/DMEM for 12 h without gAcrp and media was collected as CM-12, CM-24; Cells were stimulated with gAcrp for 8 h. After changing medium, cells were further incubated with 0.1% FBS/DMEM for 24 h without gAcrp and media was collected as CM-24.

Article Snippet: Recombinant human globular adiponectin (gAcrp) was acquired from Peprotech Inc. (Rocky Hill, NJ, USA) and 5-Chloromethyl-2, 7-dichlorodihydrofluorescein diacetate (CMH2DCFDA) from Molecular Probes (Eugene, OR).

Techniques: Expressing, Western Blot, Control, Incubation

Journal: PLoS ONE

Article Title: Globular Adiponectin Causes Tolerance to LPS-Induced TNF-α Expression via Autophagy Induction in RAW 264.7 Macrophages: Involvement of SIRT1/FoxO3A Axis

doi: 10.1371/journal.pone.0124636

Figure Lengend Snippet: (A) Cells were pretreated with gAcrp (1 μg/ml) for 24 h, followed by treatment with LPS (100 ng/ml) for additional 2 h. TNF-α mRNA level was assessed by qRT-PCR as described in materials and methods and normalized to GAPDH mRNA. Values represent fold increase compared with control cells and are expressed as mean ± S.E.M (n = 6). * P < 0.05 compared with untreated cell; # P < 0.05 compared to cells treated with LPS. (B) Cells were pretreated with Bafilomycin A1 (a selective autophagosome-lysosome inhibitor) for 2 h in the absence or presence of gAcrp (1 μg/ml) followed by stimulation with LPS (100 ng/ml) for additional 2 h. TNF-α mRNA expression was measured by qRT-PCR as described previously. Values represent fold change relative to LPS-treated cells and are presented as mean ± S.E.M. (n = 7). * P < 0.05 compared to cells treated with LPS; # P < 0.05 compared to cells treated with LPS and gAcrp. (C) Cells were pretreated with Bafilomycin A1 in the absence or presence of gAcrp (1 μg/ml) followed by stimulation with LPS (100 ng/ml) for additional 4 h. Secretion of TNF- α protein in the media was measured by ELISA as indicated in materials and methods. Values are presented as mean ± SEM. (n = 3). * P < 0.05 compared to cells treated with LPS; # P < 0.05 compared to cells treated with LPS and gAcrp. (D) (Left panel) Cells were transfected with indicated concentration of siRNA targeting LC3B or scrambled control siRNA as described in materials and methods. After 48 hours, LC3II protein expression level was determined by Western blot analysis to monitor the efficiency of gene silencing. (Right panel) Cells were transfected with siRNA targeting LC3B (25 nM) or scrambled control siRNA. After 24 h, cells were pretreated with gAcrp (1 μg/ml) for 24 h, followed by LPS treatment (100 ng/ml) for additional 2 h. TNF-α mRNA level was assessed by qRT-PCR. Values represent fold change relative to LPS-treated cells and are expressed as mean ± S.E.M. (n = 3). * P < 0.05 compared to cells treated with LPS; # P < 0.05 compared to cells treated with LPS and gAcrp. (E) Cells were transfected with siRNA targeting LC3B (25 nM) or scrambled control siRNA. After 24 h, cells were pretreated with gAcrp (1 μg/ml) for 24 h, followed by LPS treatment (100 ng/ml) for 4 h by changing the media. Secretion of TNF-α protein in the media was measured by ELISA. Values are presented as mean ± SEM. (n = 6). * P < 0.05 compared to cells treated with LPS; # P < 0.05 compared to cells treated with LPS and gAcrp. (F) Cells were pretreated with Bafilomycin A1 for 2 h, followed by treatment with LPS (100 ng/ml) for additional 2 h. TNF-α mRNA level was measured by qRT-PCR. Values represent fold change compared with control cells and expressed as mean ± S.E.M. (n = 5). * P < 0.05 compared to the control cells; # P < 0.05 compared to cells treated with LPS. (G and H) Macrophages were isolated from peritoneum of mice as described previously. Cells were pretreated with Bafilomycin A1 in the absence or presence of gAcrp and LPS essentially for the treatment of RAW 264.7 macrophages (shown in Fig 3B and C). TNF-α mRNA level was measured by RT-PCR and normalized to GAPDH mRNA (G) and the amount of TNF-α protein secreted to the media was measured by ELISA (H). Values are presented as mean ± S.E.M. (n = 3). * P < 0.05 compared with LPS; # P < 0.05 compared to cells treated with LPS and gAcrp.

Article Snippet: Recombinant human globular adiponectin (gAcrp) was acquired from Peprotech Inc. (Rocky Hill, NJ, USA) and 5-Chloromethyl-2, 7-dichlorodihydrofluorescein diacetate (CMH2DCFDA) from Molecular Probes (Eugene, OR).

Techniques: Quantitative RT-PCR, Control, Expressing, Enzyme-linked Immunosorbent Assay, Transfection, Concentration Assay, Western Blot, Isolation, Reverse Transcription Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Globular Adiponectin Causes Tolerance to LPS-Induced TNF-α Expression via Autophagy Induction in RAW 264.7 Macrophages: Involvement of SIRT1/FoxO3A Axis

doi: 10.1371/journal.pone.0124636

Figure Lengend Snippet: (A and C) Cells were pretreated with Bafilomycin A1 (10 nM) for 2 h followed by treatment with gAcrp (1 μg/ml) for additional 24 h. Cells were then treated with LPS (100 ng/ml) for 30 minutes. (B and D) Cells were transfected with siRNA targeting LC3B (25 nM) or scrambled control siRNA. After 48 hours, cells were treated with gAcrp (1 μg/ml) for 24 h, followed by treatment with LPS (100 ng/ml) for additional 30 minutes. Western blot analysis was performed to detect the level of phosphorylated p38MAPK (A, B) along with total p38MAPK, and TRAF6 (C, D) expression along with β-actin as an internal control. Representative images of three independent experiments that showed similar results are shown. Quantitative analysis of phosphorylated p38MAPK and TRAF6 expression were performed by densitometric analysis and are shown in the graph (lower panel of each figure). Values are presented as mean ± S.E.M. (n = 3). * P < 0.05 compared with LPS treated cell; # P < 0.05 compared to cells treated with LPS and gAcrp.

Article Snippet: Recombinant human globular adiponectin (gAcrp) was acquired from Peprotech Inc. (Rocky Hill, NJ, USA) and 5-Chloromethyl-2, 7-dichlorodihydrofluorescein diacetate (CMH2DCFDA) from Molecular Probes (Eugene, OR).

Techniques: Transfection, Control, Western Blot, Expressing

Journal: PLoS ONE

Article Title: Globular Adiponectin Causes Tolerance to LPS-Induced TNF-α Expression via Autophagy Induction in RAW 264.7 Macrophages: Involvement of SIRT1/FoxO3A Axis

doi: 10.1371/journal.pone.0124636

Figure Lengend Snippet: (A) Cells were treated with gAcrp (1 μg/ml) for the indicated time periods. Cytosolic and nuclear protein fractions were prepared as described in the materials and methods and the level of FoxO3A in each fraction was determined by Western blot analysis along with β-actin and lamin B1 as internal loading control for cytosolic and nuclear fractions, respectively. Quantitative analyses of foxO3A in cytosol and nucleus were performed by densitometric analysis and are shown in the graph (lower panel). Values are presented as mean ± S.E.M. * P < 0.05 compared with control cells in the cytosol fraction, # P < 0.05 compared with control cells in the nucleus fraction, respectively. (B) and (C) Cells were transfected with indicated concentration of FoxO3A siRNA or scrambled control siRNA for 48 h. The efficiency of FoxO3A silencing was measured by Western blot analysis, keeping β-actin as an internal loading control (Upper panel of figure B). Cells were transfected with siRNA targeting FoxO3A (50 nM) or scrambled control siRNA. After 48 h incubation, cells were treated with gAcrp (1 μg/ml) for 24 h and LC3II (B) or Atg5 (C) protein expression levels were determined by Western blot analysis. Representative images from three independent experiments are shown keeping β-actin as an internal loading control. Quantitative analysis of LC3II and Atg5 expression were performed by densitometric analysis and are shown in the graph (lower panel of each figure). Values are presented as mean ± S.E.M. (n = 3). * P < 0.05 compared with control; # P < 0.05 compared to cells treated with gAcrp.

Article Snippet: Recombinant human globular adiponectin (gAcrp) was acquired from Peprotech Inc. (Rocky Hill, NJ, USA) and 5-Chloromethyl-2, 7-dichlorodihydrofluorescein diacetate (CMH2DCFDA) from Molecular Probes (Eugene, OR).

Techniques: Western Blot, Control, Transfection, Concentration Assay, Incubation, Expressing

Journal: PLoS ONE

Article Title: Globular Adiponectin Causes Tolerance to LPS-Induced TNF-α Expression via Autophagy Induction in RAW 264.7 Macrophages: Involvement of SIRT1/FoxO3A Axis

doi: 10.1371/journal.pone.0124636

Figure Lengend Snippet: (A) Cells cultured in 96-well black plate were treated with different concentration of gAcrp for 24 h (left panel) or 1 μg/ml of gAcrp for different time duration (right panel). ROS production was determined using fluorometer as described previously. Data represent fold change compared to control cells and are expressed as mean ± SEM (n = 5), * P < 0.05 compared with control. (B) RAW 264.7 macrophages were treated with different concentration of gAcrp for 24 h. NADPH oxidase activity was determined by lucigenin-based assay as described in materials and methods. Values represent fold increase in compared to control cells and are expressed as mean ± S.E.M. (n = 3). * P < 0.05 compared with control cells. (C and D) Cells were pretreated with N-AC (30 mM) (C) or DPI (2.5 μM) (D) for 1 h, followed by treatment with gAcrp (1 μg/ml) for additional 24 h. LC3II protein expression level was measured by Western blot analysis as described previously. Images are representative of three independent experiments along with β-actin as internal loading control. LC3II protein expression was quantitated by densitometric analysis and is shown in the graph (lower panel) and values are presented as mean ± S.E.M. (n = 3). * P < 0.05 compared with control; # P < 0.05 compared to cells treated with gAcrp. (E and F) Cells were transiently transfected with eGFP-LC3 plasmid. After 48 h incubation, cells were pretreated with N-AC (E) or DPI (F) for 1 h followed by treatment with gAcrp for additional 24 h. GFP-LC3 dots formation was viewed with A1 Confocal Laser Microscope System as described previously. Representative images from three independent experiments that showed similar results are shown along with quantitation of LC3 dots (lower panel). Values are expressed as percentage of cells with GFP-LC3 dots obtained from at least 100 cells. * P < 0.05 compared with control; # P < 0.05 compared to cells treated with gAcrp. (G and H) Cells were pretreated with N-AC (G) or DPI (G) for 1 h followed by treatment with gAcrp (1 μg/ml) for additional 24 h. Cytosolic and nuclear protein fractions were prepared as described previously and the expression level of FoxO3A in each fraction was determined by Western blot analysis. Images are representative of three separate experiments that showed similar results along with β-actin and laminB1 as internal loading control for each fraction. Densitometric analysis was performed to quantitate protein and is shown in the graph (lower panel) and values are presented as mean ± S.E.M. (n = 2–3). * P < 0.05 compared with control; # P < 0.05 compared to cells treated with gAcrp in the cytosol and nucleus fraction.

Article Snippet: Recombinant human globular adiponectin (gAcrp) was acquired from Peprotech Inc. (Rocky Hill, NJ, USA) and 5-Chloromethyl-2, 7-dichlorodihydrofluorescein diacetate (CMH2DCFDA) from Molecular Probes (Eugene, OR).

Techniques: Cell Culture, Concentration Assay, Control, Activity Assay, Expressing, Western Blot, Transfection, Plasmid Preparation, Incubation, Microscopy, Quantitation Assay

Journal: PLoS ONE

Article Title: Globular Adiponectin Causes Tolerance to LPS-Induced TNF-α Expression via Autophagy Induction in RAW 264.7 Macrophages: Involvement of SIRT1/FoxO3A Axis

doi: 10.1371/journal.pone.0124636

Figure Lengend Snippet: (A) Cells were treated with gAcrp (1 μg/ml) for indicated time periods. SIRT1 protein expression was determined by Western blot analysis as described previously. (B and C) Cells were pretreated with N-AC (B) or DPI (C) for 1 h followed by treatment with gAcrp (1 μg/ml) for additional 8 h. Western blot analysis was performed to determine SIRT1 protein expression level. Representative images of three independent experiments are shown, keeping β-actin as an internal loading control. Quantitative analysis for SIRT1 expression was performed by densitometric analysis and presented as mean ± S.E.M. (n = 3). * P < 0.05 compared with control; # P < 0.05 compared to cells treated with gAcrp. (D) (Upper panel) Cells were transfected with indicated concentration of siRNA targeting SIRT1 or scrambled control siRNA. Gene silencing efficiency was measured by Western blot analysis. (Lower panel) After transfection with SIRT1 siRNA or scrambled siRNA, cells were treated with gAcrp (1 μg/ml) for 24 h. FoxO3A protein expression levels in cytosol and nucleus were determined by Western blot analysis along with β-actin and lamin B1 as internal loading control for cytosolic and nuclear respectively. Images are representative of three independent experiments. Densitometric analysis was done to quantitate protein and is shown in the graph (right panel) and values are presented as mean ± S.E.M. (n = 3). * P < 0.05 compared with control; # P < 0.05 compared to cells treated with gAcrp in the cytosol and nucleus fraction. (E) RAW 264.7 macrophages were treated with SIRT1 siRNA and gAcrp essentially same as above. LC3II expression level was monitored by Western blot analysis as described previously. Quantitative analysis for FoxO3A and LC3II protein expression were performed by densitometric analysis and are shown in the graph (lower panel of each figure). Values are expressed as mean ± SEM (n = 3). * P < 0.05 compared with control; # P < 0.05 compared to cells treated with gAcrp.

Article Snippet: Recombinant human globular adiponectin (gAcrp) was acquired from Peprotech Inc. (Rocky Hill, NJ, USA) and 5-Chloromethyl-2, 7-dichlorodihydrofluorescein diacetate (CMH2DCFDA) from Molecular Probes (Eugene, OR).

Techniques: Expressing, Western Blot, Control, Transfection, Concentration Assay

Journal: PLoS ONE

Article Title: Globular Adiponectin Causes Tolerance to LPS-Induced TNF-α Expression via Autophagy Induction in RAW 264.7 Macrophages: Involvement of SIRT1/FoxO3A Axis

doi: 10.1371/journal.pone.0124636

Figure Lengend Snippet: LPS treatment induces increase in TNF-α expression in macrophages via activation of TLR4 signaling, including TRAF6 induction, MKK-6 activation, p38MAPK phosphorylation and transcriptional activation of NF-κB. Treatment of macrophages with globular adiponectin causes expression of genes related with autophagy, including LC3II and Atg5, through activation of FoxO3A signaling, which is dependent on ROS production and SIRT1 expression. The autophagosome formation by gAcrp causes sequestration and degradation of TRAF6, further dampens the LPS-activated TLR4 signaling by inhibition of phosphorylation of p38MAPK, leading to the inhibition of LPS-stimulated TNF-α expression. Detailed molecular mechanism explaining how autophagy degrades TRAF6 and inhibits p38MAPK phosphorylation remained to be determined.

Article Snippet: Recombinant human globular adiponectin (gAcrp) was acquired from Peprotech Inc. (Rocky Hill, NJ, USA) and 5-Chloromethyl-2, 7-dichlorodihydrofluorescein diacetate (CMH2DCFDA) from Molecular Probes (Eugene, OR).

Techniques: Expressing, Activation Assay, Phospho-proteomics, Inhibition

Journal: PLoS ONE

Article Title: Adiponectin Signaling Regulates Lipid Production in Human Sebocytes

doi: 10.1371/journal.pone.0169824

Figure Lengend Snippet: (A) Human sebaceous glands were assayed for adiponectin receptor (AdipoR)1 and AdipoR2 by immunohistochemistry. Inset, isotype control. (B) Immunofluorescence labeling of AdipoR1 and AdipoR2 (green) in human sebocytes. Nuclei were counterstained with DAPI (blue). Inset, isotype control. (C, D) Western blotting and RT-PCR of sebocyte lysates. Human keratinocytes and fibroblasts expressing AdipoR1 and AdipoR2 were used as positive controls. Scale bars = 20 μm. Sebo, sebocytes; KC, keratinocytes; FB, fibroblasts.

Article Snippet: Recombinant human full-length adiponectin was obtained from Biobud (Seongnam, Korea).

Techniques: Immunohistochemistry, Control, Immunofluorescence, Labeling, Western Blot, Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: PLoS ONE

Article Title: Adiponectin Signaling Regulates Lipid Production in Human Sebocytes

doi: 10.1371/journal.pone.0169824

Figure Lengend Snippet: (A) The effects of adiponectin on sebocyte viability were examined by MTT assay. (B) Rate of [ 3 H]-thymidine incorporation into sebocytes treated with various doses of adiponectin, calculated as a percentage of the value in untreated cells. (c) Dose-dependent effects of adiponectin on sebocyte proliferation were examined using microscopy from low cell seeding assays. Scale bars = 400 μm. Data represent means ± SEM ( n = 6). Data were analyzed using Student’s t test (* P < 0.05).

Article Snippet: Recombinant human full-length adiponectin was obtained from Biobud (Seongnam, Korea).

Techniques: MTT Assay, Microscopy

Journal: PLoS ONE

Article Title: Adiponectin Signaling Regulates Lipid Production in Human Sebocytes

doi: 10.1371/journal.pone.0169824

Figure Lengend Snippet: (A) Detection of intracellular lipids in sebocytes treated with adiponectin using microscopy after Oil Red O and Nile red staining. Scale bars = 20 μm. (B) Lipid levels in sebocytes treated with various doses of adiponectin, calculated as percentages of the value of untreated cells. (C) Relative abundance of major lipid classes determined by thin-layer chromatography. Human sebocytes grown in the presence of [ 14 C]-acetate after treatment with adiponectin, and changes in specific lipid components such as cholesterol, triglyceride, wax ester, and squalene, were analyzed. (D) The effect of adiponectin in three-dimensional (3D) culture of human sebocytes. Sebocytes in 400 μL of medium were added to the matrigel layer (50–100 μL) adding to glass-bottom dishes. Sebocytes were treated with 200 ng/mL adiponectin and the overlay medium was replaced every 2 days during culture of the cells for 7 days. Organoid structures in 3D culture of sebocytes were stained with hematoxylin and eosin (H&E), Nile red, SREBP and epithelial membrane antigen (EMA). Scale bars = 20 μm. Data represent means ± SEM ( n = 8). Data were analyzed using Student’s t test (* P < 0.05, ** P < 0.01).

Article Snippet: Recombinant human full-length adiponectin was obtained from Biobud (Seongnam, Korea).

Techniques: Microscopy, Staining, Thin Layer Chromatography, Membrane

Journal: PLoS ONE

Article Title: Adiponectin Signaling Regulates Lipid Production in Human Sebocytes

doi: 10.1371/journal.pone.0169824

Figure Lengend Snippet: (A) After sebocytes were treated with adiponectin, whole cell lysates were prepared and analyzed by Western blotting. Blots were incubated with specific antibodies. Densitometric analyses of these protein signals were normalized relative to those for actin controls. (B) Sebocytes were transfected with control, AdipoR1 or AdipoR2-targeting siRNA and the intracellular protein levels of each signals were determined by Western blotting. (C) Sebocytes were incubated with different concentrations of compound C and protein signals were determined by Western blotting and intracellular lipid levels were measured by Oil Red O staining. Data represent means ± SEM ( n = 6). Data were analyzed by Student’s t test (* P < 0.05, ** P < 0.01, *** P < 0.001 vs . untreated cells; †P < 0.05, ‡P < 0.01 vs . adiponectin-treated cells).

Article Snippet: Recombinant human full-length adiponectin was obtained from Biobud (Seongnam, Korea).

Techniques: Western Blot, Incubation, Transfection, Control, Staining

Journal: Investigative Ophthalmology & Visual Science

Article Title: Lower Levels of Adiponectin and Its Receptor Adipor1 in the Uveal Melanomas With Monosomy-3

doi: 10.1167/iovs.61.5.12

Figure Lengend Snippet: Objective quantification of the immunohistochemical staining and the assessment of M3 status. ( a ) IHC on the paraffin sections of primary UM was performed using HRP-conjugated secondary antibodies and the HRP-green substrate, which yields a blue-green reaction product (left panel). The right panel demonstrates the negative control of the same tumor, which was incubated without the primary antibodies. Nuclei were counterstained with nuclear fast red. For each patient, light microscopy images of the entire tumor area were acquired under 200 × magnification. Image deconvolution was then performed using the Fiji software to separate the layers of nuclei, pigmentation, and IHC reaction with minimal overlap and background. The gray value of the IHC layer (as demonstrated for adiponectin) was inverted so that the regions exhibiting a stronger immunoreactivity would appear lighter and acquire higher pixel intensities compared to the background. The tumor area was then circumscribed on the original image (as demonstrated by the black lines ), and the integrated density (area × intensity) of the selected region was determined by redirecting the measurement to the inverted IHC image. This approach enabled the measurement of IHC intensity even in the samples that exhibited a very weak immunoreactivity, such as the negative control in the right panel. The mean IHC-intensities of the circumscribed areas in the positive and negative stainings were measured as 44.728 and 1.975 gray values, respectively, using this method. ( b ) Melan-A was selected as a suitable marker for the distinction of UM borders from the adjacent tissues by fluorescence microscopy. This marker was used in the subsequent Immuno-FISH assays to ensure that the nuclei that were quantified for chromosome 3 belonged to the UM cells. ( c ) Immuno-FISH for chromosome 3 was also performed on the retinal tissue of three enucleation samples as a positive control for diploid cells. Arrow indicates the autofluorescence in photoreceptor outer segments. GCL, ganglion cell layer; INL, inner nerve layer; ONL, outer nerve layer. Scale bars in ( b ) and ( c ) = 25 µm.

Article Snippet: Recombinant human adiponectin (Peprotech, Hamburg, Germany; 450-24) was reconstituted in sterile triple-distilled water.

Techniques: Immunohistochemical staining, Staining, Negative Control, Incubation, Light Microscopy, Software, Marker, Fluorescence, Microscopy, Positive Control

Journal: Investigative Ophthalmology & Visual Science

Article Title: Lower Levels of Adiponectin and Its Receptor Adipor1 in the Uveal Melanomas With Monosomy-3

doi: 10.1167/iovs.61.5.12

Figure Lengend Snippet: Adiponectin levels in the primary UM samples with regard to the M3 status. ( a ) IHC for adiponectin demonstrated a strong and relatively homogenous distribution of this protein in both the margins and the center of a primary UM, which was classified as having „low M3“ (obtained from Patient 3, male, 67 years). In contrast, the “high M3” tumor (derived from patient 14, female, 48 years) exhibited significantly less adiponectin in the periphery, which decreased further toward the center. The adiponectin-positive cells in the center of this latter tumor, which constituted less than 10% of the cell population, were mainly detected in the vicinity of blood vessels (indicated by the black arrows ). Patient 3 received stereotactic radiotherapy 17 months before undergoing endoresection, whereas patient 14 underwent enucleation without irradiation. Patient 14 developed liver metastases within 2 years, whereas no metastases or extraocular growth were detected in Patient 3 during the follow-up time of 9 years. ( b ) Immuno-FISH assay demonstrating the copy number of chromosome 3 in the Melan-A positive cells of these tumors. Note the lower number of chromosome 3 signals despite the higher nuclear density in the “high M3” tumor. Examples of cells with M3 are indicated by the white arrows in both tumors. ( c ) Quantification of adiponectin in the entire tumor area with respect to the M3 status. Tumors with a higher percentage of M3 positive cells (n = 16) had significantly lower levels of adiponectin compared to the low M3 tumors (n = 22) as determined by logistic regression analysis. ( d ) Patients with a higher percentage of M3 in their CMC (n = 15) also had significantly less adiponectin in their primary tumor compared with the patients with lower levels of M3-positive CMC (n = 16) as determined by logistic regression. ( e ) Examples of some of the CMC detected in patient 14 at the time of diagnosis with primary UM. Arrows indicate the immunobeads. No CMC were detected in patient 3. Scale bars in ( b ) and ( e ) = 10 µm. MCSP, Melanoma-associated chondroitin sulfate proteoglycan.

Article Snippet: Recombinant human adiponectin (Peprotech, Hamburg, Germany; 450-24) was reconstituted in sterile triple-distilled water.

Techniques: Derivative Assay, Irradiation, Biomarker Discovery

Journal: Investigative Ophthalmology & Visual Science

Article Title: Lower Levels of Adiponectin and Its Receptor Adipor1 in the Uveal Melanomas With Monosomy-3

doi: 10.1167/iovs.61.5.12

Figure Lengend Snippet: Expression of Adipor1 in primary UM samples with regard to the M3 status. ( a ) The irradiated, low M3 tumor of patient 3 exhibited a strong and mainly homogenous expression of Adipor1 in both the periphery and center. In contrast, Adipor1 was detected at significantly weaker levels in the periphery and was almost absent in the center of the nonirradiated, high M3 tumor of patient 14. Arrow indicates stronger Adipor1 expression in the proximity of a blood vessel in the latter tumor. Quantification of Adipor1 intensity in the entire tumor area demonstrated significantly lower levels of this protein in the patients who had a high extent of M3 in their primary tumor ( b ), a higher number of CMC ( c ), and a higher percentage of CMC with M3 ( d ). Adipor1 expression correlated positively with the adiponectin levels in the corresponding primary UM ( e ). The P values in ( b – e ) were determined by logistic regression analysis.

Article Snippet: Recombinant human adiponectin (Peprotech, Hamburg, Germany; 450-24) was reconstituted in sterile triple-distilled water.

Techniques: Expressing, Irradiation

Journal: Investigative Ophthalmology & Visual Science

Article Title: Lower Levels of Adiponectin and Its Receptor Adipor1 in the Uveal Melanomas With Monosomy-3

doi: 10.1167/iovs.61.5.12

Figure Lengend Snippet: Validation of the differential protein levels of adiponectin and Adipor1 with regard to the M3 status by immunoblotting. Freshly frozen tissue was available from two samples that were classified as having low versus high M3 (patients 5 and 6; female; age: 53 and 69 years, respectively). The remaining halves of the tissues were fixed in formalin and processed for immunostaining and immuno-FISH. Both patients underwent irradiation before the acquisition of the tumor samples. ( A ) IHC for adiponectin and Adipor1 demonstrated a reduction in the levels of both proteins in the high M3 sample. Arrows indicate several cells with M3 detected by immuno-FISH. Immunoblotting of the lysates of these tumors also revealed the stronger presence of ( B ) adiponectin and ( C ) Adipor1 in the low M3 sample. Expression of Melan-A was analyzed as a positive control for the abundance of melanoma cells in the tissue lysates. Patient 6 with the high M3 sample developed metastases within 5 years, whereas no metastases were detected in patient 5 with the low M3-tumor during the follow-up time of 8 years. Scale bar = 25 µm.

Article Snippet: Recombinant human adiponectin (Peprotech, Hamburg, Germany; 450-24) was reconstituted in sterile triple-distilled water.

Techniques: Biomarker Discovery, Western Blot, Immunostaining, Irradiation, Expressing, Positive Control

Journal: Investigative Ophthalmology & Visual Science

Article Title: Lower Levels of Adiponectin and Its Receptor Adipor1 in the Uveal Melanomas With Monosomy-3

doi: 10.1167/iovs.61.5.12

Figure Lengend Snippet: Expression of adiponectin and Adipor1 with regard to the BAP1 levels in the primary tumor of our UM patients. The immunoreactivity for BAP1 in the ( A ) cytoplasm and ( B ) nucleus was graded at a scale of 0–3 (0: positive staining in less than 10% of cells; 1: positive staining in 11%–33% of cells; 2: positive staining in 34%–66% of cells; 3: positive staining in more than 67% of cells). Images were acquired under 200x magnification. The immunoreactivity for ( C ) adiponectin and ( D ) Adipor1 was significantly reduced in the tumors with lower cytoplasmic levels of BAP1 (n = 14 and n = 23 samples with low versus high BAP1, respectively; Mann-Whitney U test).

Article Snippet: Recombinant human adiponectin (Peprotech, Hamburg, Germany; 450-24) was reconstituted in sterile triple-distilled water.

Techniques: Expressing, Staining, MANN-WHITNEY

Journal: Investigative Ophthalmology & Visual Science

Article Title: Lower Levels of Adiponectin and Its Receptor Adipor1 in the Uveal Melanomas With Monosomy-3

doi: 10.1167/iovs.61.5.12

Figure Lengend Snippet: Association of the Metastases/Extraocular Growth With Clinical Parameters and Adiponectin/Adipor1 Protein Levels (Follow-Up Time: 2–9 Years)

Article Snippet: Recombinant human adiponectin (Peprotech, Hamburg, Germany; 450-24) was reconstituted in sterile triple-distilled water.

Techniques: Biomarker Discovery, Irradiation

Journal: Investigative Ophthalmology & Visual Science

Article Title: Lower Levels of Adiponectin and Its Receptor Adipor1 in the Uveal Melanomas With Monosomy-3

doi: 10.1167/iovs.61.5.12

Figure Lengend Snippet: Expression of the major genes involved in adiponectin-mediated signaling in the UM cohort of the TCGA study (n = 80 patients). Each column represents a tumor sample, which was clustered according to the copy number of chromosome 3p and 3q ( red : normal, blue : loss) in the uppermost two rows. Tumors with the loss of both 3p and 3q were considered as having M3, whereas these data were incomplete for n = 3 samples. The copy number of chromosomes 1q, 9q, and 12, which harbor several of the genes of interest, as well as the survival status are also demonstrated in the upper group of rows, whereas the middle rows indicate the presence of genetic alterations. The lower rows construct the expression heatmap, which was generated from the mRNA z-scores, with blue and red representing mRNA levels that were up to three standard deviations lower or higher than the mean, respectively, whereas black indicates an expression at the mean. The loci of the analyzed genes are indicated in parentheses next to the gene symbol in the heatmap. Expression of BAP1 was included as a reference.

Article Snippet: Recombinant human adiponectin (Peprotech, Hamburg, Germany; 450-24) was reconstituted in sterile triple-distilled water.

Techniques: Expressing, Construct, Generated

Journal: Investigative Ophthalmology & Visual Science

Article Title: Lower Levels of Adiponectin and Its Receptor Adipor1 in the Uveal Melanomas With Monosomy-3

doi: 10.1167/iovs.61.5.12

Figure Lengend Snippet: Expression of adiponectin and its receptors in the cultured human choroidal melanocytes (hCM) and UM cells. ( a ) Culture purity was evaluated by the levels of melanocyte- and melanoma-specific markers. The hCM1 cells were derived from the choroid of a patient without an ocular tumor, whereas the hCM2 cells were obtained from the tumor-free choroidal region of a patient with posterior UM. The UM1 and UM2 cells were isolated from the primary tumors of patients 19 and 14, respectively, in our study. The donor of UM1 cells (male, 82 years) had posterior UM which was classified as a high M3 tumor. UM2 cells were derived from the high M3 tumor of the patient (female, 48 years), who was referred to in the and , as well. ( b ) Expression of adiponectin, as well as its receptors Adipor1 and Adipor2 in the cells grown in normal medium with 10% FBS. Expression of the receptors on the cell surface was analyzed by immunocytochemistry on non-permeabilized cells. Adipor1 could be detected on all the cell types whereas the expression of Adipor2 was considerably stronger in the UM cultures. Scale bar = 50 µm. ( c ) Immunoblotting under non-reducing conditions demonstrated the expression of adiponectin in the whole lysates of Mel-270 and OMM-2.5 cells that were either grown in normal medium with 10% FBS or serum-deprived for 2 days. The total protein loading in the lanes was detected by stain-free gel imaging. ( d ) Immunocytochemical analysis of adiponectin levels in the Mel-270 and OMM-2.5 cells that were serum-deprived for 2 days. The negative controls were incubated without the primary antibody. Scale bar = 25 µm.

Article Snippet: Recombinant human adiponectin (Peprotech, Hamburg, Germany; 450-24) was reconstituted in sterile triple-distilled water.

Techniques: Expressing, Cell Culture, Derivative Assay, Isolation, Immunocytochemistry, Western Blot, Staining, Imaging, Incubation

Journal: Investigative Ophthalmology & Visual Science

Article Title: Lower Levels of Adiponectin and Its Receptor Adipor1 in the Uveal Melanomas With Monosomy-3

doi: 10.1167/iovs.61.5.12

Figure Lengend Snippet: Basal adiponectin levels in the cultured UM cells with M3 versus disomy-3. ( a ) Immuno-FISH was performed for the codetection of adiponectin and chromosome 3 on the cytospins of intact UM1 and UM2 cells (both at passage 2). Images presented were acquired from the UM1 cells. Notice the larger nucleolar area (which appear darker in the nuclear stainings and were marked by the white lines ) in the cells with M3. ( b ) The mean gray value of the adiponectin staining was quantified by circumscribing the cytoplasmic region (excluding the nuclei) in a total of 112–180 nonoverlapping UM2 and UM1 cells, respectively, with clear signals for chromosome 3. The number of M3-positive cells that could be quantified was 43–69 for the UM2 and UM1 cells, respectively. The intensity of the cells with disomy-3 was taken as 100%. Data represent the mean ± standard deviation of the measurements for the UM1 and UM2 cells. P value was determined by two-sided t -test assuming equal variance.

Article Snippet: Recombinant human adiponectin (Peprotech, Hamburg, Germany; 450-24) was reconstituted in sterile triple-distilled water.

Techniques: Cell Culture, Staining, Standard Deviation

Journal: Investigative Ophthalmology & Visual Science

Article Title: Lower Levels of Adiponectin and Its Receptor Adipor1 in the Uveal Melanomas With Monosomy-3

doi: 10.1167/iovs.61.5.12

Figure Lengend Snippet: Adiponectin induces a more quiescent phenotype in the UM cells by suppressing proliferation and energy production. ( a ) Immunostaining for the proliferation marker Ki-67 on the Mel-270 and OMM-2.5 cells after a 1-day incubation in normal medium with 10% FBS, serum-free medium (no FBS), or serum-free medium with 30 µg/ml Adiponectin (no FBS + A). Scale bar = 50 µm. ( b ) Quantification of the Ki-67 immunostaining was performed on a minimum of 205 nuclei per group. Cells with a mean Ki-67 intensity that was above the cutoff value of negative cells were defined as being positive. Data represent the mean ± SEM of n = 3 independent experiments. * P < 0.05 compared to the corresponding no FBS group as determined by the two-sided t -test. ( c ) YAP-immunostaining demonstrating a gradual decline in the levels of this protein in response to serum deprivation ± adiponectin. The intensity of the nuclear staining also underwent a progressive decrease, suggesting the impairment of DNA duplication. Scale bar = 25 µm. ( d ) Immunoblotting for Ki-67 and YAP in the whole cell lysates after a 1-day exposure to the indicated treatments. Images are representative of n = 3 experiments. ( e ) Silver staining of nucleolar organizer regions (AgNOR, indicated by the arrow ), demonstrating the reduction in the number and area of the nucleolar structures in response to serum deprivation + Adiponectin. Scale bar = 25 µm. ( f ) Quantification of the AgNOR area. Data represent the mean ± SEM of n = 3 experiments with a minimum of n = 202 quantified nuclei per group. * P < 0.05 compared to the corresponding no FBS group as determined by the two-sided t -test. ( g ) Cell-cycle analysis after a 1-day exposure to the indicated treatments. Adiponectin could mainly impair the progression of UM cells into the G2/M phase and induce a slight but significant accumulation of the cells in the Sub G1 phase. The Mel-270 and OMM-2.5 cells also differed in their cell-cycle rates, with the former cell type apparently requiring more time for the G2/M phase, as demonstrated by the higher percentage of untreated Mel-270 cells in the G2/M phase and the lower cell density of this group compared with the untreated OMM-2.5 cells in panel ( a ). Data represent the mean ± SEM of n = 3 independent experiments. * P < 0.05 compared to the no FBS group of the corresponding cell as determined by the two-sided t -test. ( h ) ATP levels in the serum deprived cells treated with or without adiponectin for 8 to 10 hours as measured by a luminescence assay. The values of untreated cells were taken as 100%. Data represent the mean ± SEM of n = 3 independent experiments with triplicate wells for each group. * P < 0.05 compared to the no FBS group of the corresponding cell as determined by the two-sided t -test.

Article Snippet: Recombinant human adiponectin (Peprotech, Hamburg, Germany; 450-24) was reconstituted in sterile triple-distilled water.

Techniques: Immunostaining, Marker, Incubation, Staining, Western Blot, Silver Staining, Cell Cycle Assay, Luminescence Assay

Journal: PLoS ONE

Article Title: A Saccharomyces cerevisiae Assay System to Investigate Ligand/AdipoR1 Interactions That Lead to Cellular Signaling

doi: 10.1371/journal.pone.0065454

Figure Lengend Snippet: (A) The components of the AdipoR1 signaling pathway of mammalian cells are shown in black font and their S. cerevisiae homologs, if known are shown in red font. In mammalian cells, adaptor protein containing pleckstrin homology domain, phosphotyrosine binding domain and leucine zipper motif 1 (APPL1) interacts directly with AdipoR1. Interaction of adiponectin (ADPN) with AdipoR1 stimulates AdipoR1-APPL1 interaction. This results in release of Ca 2+ from the ER to the cytosol and also increases export of LKB1 kinase from the nucleus. Influx of extracellular Ca 2+ to the cytosol is also stimulated by adiponectin, although the mechanism by which this occurs remains to be clarified. Increase in cytosolic Ca 2+ concentration activates Ca 2+ /calmodulin-dependent protein kinase kinase (CaMKK) which in turn activates AMP activated protein kinase (AMPK) by phosphorylating its α subunit. However, phosphorylation of AMPK α subunit by the cytosol-localized kinase LKB1 is the major pathway for activation of AMPK. Adiponectin-AdipoR1 interaction also increases cellular ceramidase activity which in turn leads to phosphorylation of AMPKα subunit. The details of this pathway are not clear yet. Activation of AMPK is required for many of the anti-diabetic and anti-atherosclerotic effects of adiponectin. In vascular endothelial cells, interaction of AdipoR1 with adiponectin activates protein kinase A (PKA) which has the effect of lowering accumulation of reactive oxygen species (ROS) and thereby reducing inflammation. (B) Subunit structure of S. cerevisiae AMPK (ScAMPK). Like mammalian AMPK, ScAMPK is a trimer composed of an α, β, and γ subunit. The genes encoding the β subunit isoforms as well as the sole α and γ subunits are indicated. (C) Components of the IZH2 -mediated signaling pathways in S. cerevisiae are shown in red font. Interaction of IZH2 (homolog of AdipoRs) with osmotin (OSM) activates PKA via a RAS2-cAMP pathway. Overexpression of IZH2 , overexpression of AdipoR1 , treatment of S. cerevisiae cells expressing AdipoR1 with adiponectin and treatment of S. cerevisiae cells expressing various levels of IZH2 with thaumatin (THN, a homolog of osmotin) has been shown to activate PKA by increasing cellular ceramidase activity. Activation of PKA leads to decreased transcription from a stress responsive promoter element ( STRE ) and increased cellular ROS content. Activated PKA promotes export of ScAMPK from the nucleus which leads to decreased transcription from the ferroxidase (FET3 ) promoter. Activated PKA also represses FET3 transcription via the stress-responsive transcription factors MSN2/4. Mutational analyses show that genes encoding the APPL1-lke protein Sip3, the LKB1-like protein Sak1, the ScAMPK β subunit Sip1 and the ScAMPK γ subunit Snf4 are components of the pathway leading from IZH2 (or AdipoR1 ) to FET3 repression in S. cerevisiae .

Article Snippet: Unless specified otherwise, the pure recombinant Escherichia coli expressed N-terminal (His) 6 -tagged full length human adiponectin used in these studies was the gift of Dr. A. Sharkhuu (KAUST, Saudi Arabia) or Dr.

Techniques: Binding Assay, Concentration Assay, Phospho-proteomics, Activation Assay, Activity Assay, Protein-Protein interactions, Over Expression, Expressing

Journal: PLoS ONE

Article Title: A Saccharomyces cerevisiae Assay System to Investigate Ligand/AdipoR1 Interactions That Lead to Cellular Signaling

doi: 10.1371/journal.pone.0065454

Figure Lengend Snippet: Cells of strain BY4741carrying pESC-URA-CLuc-AdipoR1-APPL1-NLuc were grown for 16 h at 30°C in selective minimal medium at the indicated galactose concentrations, treated for 4 h at 30°C with the indicated test compounds and then assayed for Luc activity. (A) Imaging of Luc activity. (B) Quantitative measurement of Luc activity as a function of osmotin concentration. A representative image of relative Luc activity at the different osmotin concentrations is shown for each galactose concentration. Data represent the means ± SD from three experiments with triplicate samples. For each galactose concentration, significant differences by a Student’s t-test between osmotin treated samples and untreated control are indicated by asterisks. Symbols: PBS, 1/8 X PBS; f-ADPN, bacterially expressed full length adiponectin; BSA, bovine serum albumin, OSM, osmotin; g-ADPN, bacterially expressed globular adiponectin; **, p <0.01; ***, p <0.001.

Article Snippet: Unless specified otherwise, the pure recombinant Escherichia coli expressed N-terminal (His) 6 -tagged full length human adiponectin used in these studies was the gift of Dr. A. Sharkhuu (KAUST, Saudi Arabia) or Dr.

Techniques: Activity Assay, Imaging, Concentration Assay, Control

Journal: PLoS ONE

Article Title: A Saccharomyces cerevisiae Assay System to Investigate Ligand/AdipoR1 Interactions That Lead to Cellular Signaling

doi: 10.1371/journal.pone.0065454

Figure Lengend Snippet: (A) Cell lysates (100 µg protein) of strain BWG1-7a and an isogenic Δsnf1::Kan_MX line were fractionated by 10% SDS-PAGE. Shown are blots probed with anti-phospho-AMPK(Thr-172) antibody. The expected size of Snf1p is 72 kDa. (B, C) Cells (about 10 8 /mL) of strain BWG1-7a Δizh2::Kan_MX transformed with p426GPD (Vec), p426GPD- AdipoR1 (pAdipoR1) or p426GPD- AdipoR2 (pAdipoR2) were treated with indicated osmotin and adiponectin concentrations for 30 min at 30°C in YPD. Aliquots were withdrawn for viable counts determination before the cell lysates were prepared for analysis by10% SDS-PAGE. Shown in the top panels are blots probed first with phospho-AMPK(Thr-172) antibody, then stripped and probed with actin antibody (100 µg total protein per lane). Shown in the middle panels are relative band intensities in the depicted gels. ‘Relative band intensity’ was defined as the ratio of the intensity of phospho-Snf1p signal to actin signal for each lane when the value of this ratio for the corresponding untreated Vec sample was arbitrarily assigned the value 1.0. Shown in the bottom panels are viable counts in each sample at the end of the treatments. The experiments were performed twice with comparable results and the results of one experiment are shown.

Article Snippet: Unless specified otherwise, the pure recombinant Escherichia coli expressed N-terminal (His) 6 -tagged full length human adiponectin used in these studies was the gift of Dr. A. Sharkhuu (KAUST, Saudi Arabia) or Dr.

Techniques: SDS Page, Transformation Assay