recombinant human globular adiponectin Search Results


recombinant human gadiponectin/gacrp30 protein, cf  (Bio-Techne corporation)
90
Bio-Techne corporation recombinant human gadiponectin/gacrp30 protein, cf
Recombinant Human Gadiponectin/Gacrp30 Protein, Cf, supplied by Bio-Techne corporation, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/recombinant human gadiponectin/gacrp30 protein, cf/product/Bio-Techne corporation
Average 90 stars, based on 1 article reviews
recombinant human gadiponectin/gacrp30 protein, cf - by Bioz Stars, 2026-03
90/100 stars
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recombinant human globular adiponectin (gacrp)  (PeproTech)
90
PeproTech recombinant human globular adiponectin (gacrp)
(A and B) RAW 264.7 macrophages were treated with <t>gAcrp</t> (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.
Recombinant Human Globular Adiponectin (Gacrp), supplied by PeproTech, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/recombinant human globular adiponectin (gacrp)/product/PeproTech
Average 90 stars, based on 1 article reviews
recombinant human globular adiponectin (gacrp) - by Bioz Stars, 2026-03
90/100 stars
  Buy from Supplier
human or mouse recombinant globular adiponectin (rgadpn)  (MBL International)
90
MBL International human or mouse recombinant globular adiponectin (rgadpn)
(A and B) RAW 264.7 macrophages were treated with <t>gAcrp</t> (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.
Human Or Mouse Recombinant Globular Adiponectin (Rgadpn), supplied by MBL International, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/human or mouse recombinant globular adiponectin (rgadpn)/product/MBL International
Average 90 stars, based on 1 article reviews
human or mouse recombinant globular adiponectin (rgadpn) - by Bioz Stars, 2026-03
90/100 stars
  Buy from Supplier

Image Search Results


(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.

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

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.

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

(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.

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

(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.

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

(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.

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

(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.

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

(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.

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

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.

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