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recombinant human globular adiponectin (gacrp; #450-21) (PeproTech)

Name: recombinant human globular adiponectin (gacrp; #450-21)
Brand: PeproTech
Rating: 90 (1 reviews)

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PeproTech recombinant human globular adiponectin (gacrp; #450-21)
Recombinant Human Globular Adiponectin (Gacrp; #450 21), 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
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Average 90 stars, based on 1 article reviews

recombinant human globular adiponectin (gacrp; #450-21) - by Bioz Stars, 2026-03

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Suppression of the inflammasome activation by globular adiponectin in breast and hepatic cancer cells. ( A – H ) MCF-7 breast cancer cells were treated with gAcrp (1 µg/mL) for the indicated time duration ( A , C , E , G ) or treated with gAcrp at different concentrations for 24 h ( B , D , F , H ). Total protein lysates were immunoblotted for IL-1β ( A , B ), Caspase-1 ( C , D ), NOD-like receptor pyrin domain-containing protein 3 (NLRP3) ( E , F ), or the apoptosis-associated speck-like protein containing a CARD (ASC) ( G , H ). ( I ) MCF-7 cells were treated with gAcrp (1 µg/mL) for the indicated time periods before sequentially incubated with antibodies for ASC (green) and 4′,6-diamidino-2-phenylindole (DAPI) (blue). ASC speck formation was indicated by white arrows. Representative images from three independent experiments were presented along with the quantitation of ASC speck in the right panel. Values are expressed as percentage of the cells presenting the ASC dots with respect to DAPI by using Image Inside Software version 2.32. Scale bar: 5 µm. ( J – M ) HepG2 hepatic cancer cells were incubated with gAcrp (1 µg/mL) for time durations as indicated. Western blot analyses were performed for the measurement of IL-1β (J), caspase-1 (K), NLRP3 (L), and ASC (M). For Western blot experiments, representative images from three independent experiments are presented. β-actin was used as a loading control. Bar diagrams show the relative band intensity of the target proteins compared to β-actin or the respective proform, determined by densitometric analysis. Values are presented as the fold change compared with the control cells and are expressed as mean ± standard error of mean (SEM). * denotes p < 0.05 compared with control cells.

Journal: Cancers

Article Title: Globular Adiponectin Inhibits Breast Cancer Cell Growth through Modulation of Inflammasome Activation: Critical Role of Sestrin2 and AMPK Signaling

doi: 10.3390/cancers12030613

Figure Lengend Snippet: Suppression of the inflammasome activation by globular adiponectin in breast and hepatic cancer cells. ( A – H ) MCF-7 breast cancer cells were treated with gAcrp (1 µg/mL) for the indicated time duration ( A , C , E , G ) or treated with gAcrp at different concentrations for 24 h ( B , D , F , H ). Total protein lysates were immunoblotted for IL-1β ( A , B ), Caspase-1 ( C , D ), NOD-like receptor pyrin domain-containing protein 3 (NLRP3) ( E , F ), or the apoptosis-associated speck-like protein containing a CARD (ASC) ( G , H ). ( I ) MCF-7 cells were treated with gAcrp (1 µg/mL) for the indicated time periods before sequentially incubated with antibodies for ASC (green) and 4′,6-diamidino-2-phenylindole (DAPI) (blue). ASC speck formation was indicated by white arrows. Representative images from three independent experiments were presented along with the quantitation of ASC speck in the right panel. Values are expressed as percentage of the cells presenting the ASC dots with respect to DAPI by using Image Inside Software version 2.32. Scale bar: 5 µm. ( J – M ) HepG2 hepatic cancer cells were incubated with gAcrp (1 µg/mL) for time durations as indicated. Western blot analyses were performed for the measurement of IL-1β (J), caspase-1 (K), NLRP3 (L), and ASC (M). For Western blot experiments, representative images from three independent experiments are presented. β-actin was used as a loading control. Bar diagrams show the relative band intensity of the target proteins compared to β-actin or the respective proform, determined by densitometric analysis. Values are presented as the fold change compared with the control cells and are expressed as mean ± standard error of mean (SEM). * denotes p < 0.05 compared with control cells.

Article Snippet: Recombinant human globular adiponectin (gAcrp) was purchased from PeproTech Inc. (Rocky Hill, NJ, USA).

Techniques: Activation Assay, Incubation, Quantitation Assay, Software, Western Blot, Control

Suppression of ER stress by globular adiponectin and its implication in the modulation of inflammasomes activation in breast cancer cells. ( A – C ) MCF-7 cells were treated with gAcrp (1 µg/mL) for the indicated time duration. Expression levels of phospho- and total protein kinase RNA-like endoplasmic reticulum kinase (PERK) ( A ), phospho- and total eukaryotic translation initiation factor 2A (eIF2α) (B), and C/EBP homologous protein (CHOP) were determined by Western blot analysis. ( D – G ) MCF-7 cells were incubated with the indicated concentrations of tauroursodeoxycholic acid (TUDCA) ( D , E ) or tunicamycin ( F , G ) for 24 h or 12 h, respectively. Immunoblot analysis was carried out for determining the levels of interleukin-1β (IL-1β) and caspase-1. For all the Western blot analyses, the expression level of the target genes was estimated by densitometric analysis and is shown in the lower panel. Values represent fold change in comparison to the control group after being normalized to β-actin and are expressed as mean ± standard error of mean (SEM), n = 3. * denotes p < 0.05 compared with control cells.

Journal: Cancers

Article Title: Globular Adiponectin Inhibits Breast Cancer Cell Growth through Modulation of Inflammasome Activation: Critical Role of Sestrin2 and AMPK Signaling

doi: 10.3390/cancers12030613

Figure Lengend Snippet: Suppression of ER stress by globular adiponectin and its implication in the modulation of inflammasomes activation in breast cancer cells. ( A – C ) MCF-7 cells were treated with gAcrp (1 µg/mL) for the indicated time duration. Expression levels of phospho- and total protein kinase RNA-like endoplasmic reticulum kinase (PERK) ( A ), phospho- and total eukaryotic translation initiation factor 2A (eIF2α) (B), and C/EBP homologous protein (CHOP) were determined by Western blot analysis. ( D – G ) MCF-7 cells were incubated with the indicated concentrations of tauroursodeoxycholic acid (TUDCA) ( D , E ) or tunicamycin ( F , G ) for 24 h or 12 h, respectively. Immunoblot analysis was carried out for determining the levels of interleukin-1β (IL-1β) and caspase-1. For all the Western blot analyses, the expression level of the target genes was estimated by densitometric analysis and is shown in the lower panel. Values represent fold change in comparison to the control group after being normalized to β-actin and are expressed as mean ± standard error of mean (SEM), n = 3. * denotes p < 0.05 compared with control cells.

Article Snippet: Recombinant human globular adiponectin (gAcrp) was purchased from PeproTech Inc. (Rocky Hill, NJ, USA).

Techniques: Activation Assay, Expressing, Western Blot, Incubation, Comparison, Control

Crucial role of AMP-activated protein kinase (AMPK) signaling in the modulation of endoplasmic reticulum (ER) stress and inflammasomes by globular adiponectin. ( A ) MCF-7 cells were treated with gAcrp (1 µg/mL) for the indicated time periods. The expression levels of AMPKα were measured by Western blot analysis. ( B , C ) MCF-7 cells were pretreated with compound C (1 µM), a pharmacological inhibitor of AMPK, for 1h followed by further treatment with gAcrp (1 µg/mL) for additional 24 h. Total protein lysates were prepared and used for immunoblotting analysis for the measurement of interleukin-1β (IL-1β) ( B ) and Caspase-1 ( C ). ( D ) After pretreatment with compound C for 1 h, MCF-7 cells were incubated with gAcrp (1 µg/mL) for additional 1 h and further incubated with antibodies for specific ASC (green) and DAPI (blue). Scale bar: 5µm. ( E – I ) MCF-7 cells were transfected with siRNA targeting AMPKα or control scrambled siRNA. Gene silencing efficiency of AMPKαwas monitored by Western blot analysis (Upper panel in E). After transient gene silencing of AMPKα, cells were treated with gAcrp for 24h ( E , F ) or 1 h ( G – I ). IL-1β (E), Caspase-1 ( F ), protein kinase RNA-like endoplasmic reticulum kinase (PERK) (G), Eukaryotic translation initiation factor 2A (eIF2α) ( H ), and C/EBP homologous protein (CHOP) ( I ) expression levels were determined by Western blot analysis. Values are presented as the fold change compared with the control cells and are expressed as mean± standard error of mean (SEM), n = 3. * denotes p < 0.05 compared to control cells, # denotes p < 0.05 compared with the cells treated with globular adiponectin but not pretreated with compound C ( B , C ) or not transfected with siRNA ( E – I ).

Journal: Cancers

Article Title: Globular Adiponectin Inhibits Breast Cancer Cell Growth through Modulation of Inflammasome Activation: Critical Role of Sestrin2 and AMPK Signaling

doi: 10.3390/cancers12030613

Figure Lengend Snippet: Crucial role of AMP-activated protein kinase (AMPK) signaling in the modulation of endoplasmic reticulum (ER) stress and inflammasomes by globular adiponectin. ( A ) MCF-7 cells were treated with gAcrp (1 µg/mL) for the indicated time periods. The expression levels of AMPKα were measured by Western blot analysis. ( B , C ) MCF-7 cells were pretreated with compound C (1 µM), a pharmacological inhibitor of AMPK, for 1h followed by further treatment with gAcrp (1 µg/mL) for additional 24 h. Total protein lysates were prepared and used for immunoblotting analysis for the measurement of interleukin-1β (IL-1β) ( B ) and Caspase-1 ( C ). ( D ) After pretreatment with compound C for 1 h, MCF-7 cells were incubated with gAcrp (1 µg/mL) for additional 1 h and further incubated with antibodies for specific ASC (green) and DAPI (blue). Scale bar: 5µm. ( E – I ) MCF-7 cells were transfected with siRNA targeting AMPKα or control scrambled siRNA. Gene silencing efficiency of AMPKαwas monitored by Western blot analysis (Upper panel in E). After transient gene silencing of AMPKα, cells were treated with gAcrp for 24h ( E , F ) or 1 h ( G – I ). IL-1β (E), Caspase-1 ( F ), protein kinase RNA-like endoplasmic reticulum kinase (PERK) (G), Eukaryotic translation initiation factor 2A (eIF2α) ( H ), and C/EBP homologous protein (CHOP) ( I ) expression levels were determined by Western blot analysis. Values are presented as the fold change compared with the control cells and are expressed as mean± standard error of mean (SEM), n = 3. * denotes p < 0.05 compared to control cells, # denotes p < 0.05 compared with the cells treated with globular adiponectin but not pretreated with compound C ( B , C ) or not transfected with siRNA ( E – I ).

Article Snippet: Recombinant human globular adiponectin (gAcrp) was purchased from PeproTech Inc. (Rocky Hill, NJ, USA).

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

Role of sestrin2 (SESN2) induction in AMP-activated protein kinase (AMPK) phosphorylation, endoplasmic reticulum (ER) stress amelioration, and inflammasome inhibition by globular adiponectin in breast cancer cells. ( A ) MCF-7 cells were treated with gAcrp (1 µg/mL) for the indicated time periods. The expression level of SESN2 was determined by Western blot analysis. ( B ) MCF-7 cells were transiently transfected with siRNA targeting AMPKα and the gene silencing efficiency was monitored after 36 h using immunoblotting analysis (upper panel). After transfection, MCF-7 cells were further incubated with gAcrp (1 µg/mL) for 1 h and the expression of AMPK was examined (lower panel). ( C ) MCF-7 cells were treated with gAcrp (1 µg/mL) for 30 min. The SESN2-associated protein levels of AMPK and LKB-1 were determined by Immunoprecipitation assay. ( D ) After transfection with SESN2 siRNA, MCF-7 cells were further incubated with gAcrp (1 µg/mL) for 30 min. AMPKα amount associated with liver kinase B1 (LKB-1) was measured by immunoprecipitation assay. ( E – I ) MCF-7 cells were transfected with siRNA targeting SESN2 or control scrambled siRNA followed by treatment with gAcrp (1 µg/mL) for 1 h (G,H) or 24 h ( E , F , I ). Total protein lysates were immunoblotted for interleukin-1β (IL-1β) (E), Caspase-1 ( F ), the protein kinase RNA-like endoplasmic reticulum kinase (PERK) ( G ), Eukaryotic translation initiation factor 2A (eIF2α) ( H ), and C/EBP Homologous Protein (CHOP) ( I ). Values represent the fold change relative to the control cells and are expressed as mean± standard error of mean, n = 3. * denotes p < 0.05 compared to control cells, # denotes p < 0.05 compared with the cells treated with gAcrp but not transfected with siRNA.

Journal: Cancers

Article Title: Globular Adiponectin Inhibits Breast Cancer Cell Growth through Modulation of Inflammasome Activation: Critical Role of Sestrin2 and AMPK Signaling

doi: 10.3390/cancers12030613

Figure Lengend Snippet: Role of sestrin2 (SESN2) induction in AMP-activated protein kinase (AMPK) phosphorylation, endoplasmic reticulum (ER) stress amelioration, and inflammasome inhibition by globular adiponectin in breast cancer cells. ( A ) MCF-7 cells were treated with gAcrp (1 µg/mL) for the indicated time periods. The expression level of SESN2 was determined by Western blot analysis. ( B ) MCF-7 cells were transiently transfected with siRNA targeting AMPKα and the gene silencing efficiency was monitored after 36 h using immunoblotting analysis (upper panel). After transfection, MCF-7 cells were further incubated with gAcrp (1 µg/mL) for 1 h and the expression of AMPK was examined (lower panel). ( C ) MCF-7 cells were treated with gAcrp (1 µg/mL) for 30 min. The SESN2-associated protein levels of AMPK and LKB-1 were determined by Immunoprecipitation assay. ( D ) After transfection with SESN2 siRNA, MCF-7 cells were further incubated with gAcrp (1 µg/mL) for 30 min. AMPKα amount associated with liver kinase B1 (LKB-1) was measured by immunoprecipitation assay. ( E – I ) MCF-7 cells were transfected with siRNA targeting SESN2 or control scrambled siRNA followed by treatment with gAcrp (1 µg/mL) for 1 h (G,H) or 24 h ( E , F , I ). Total protein lysates were immunoblotted for interleukin-1β (IL-1β) (E), Caspase-1 ( F ), the protein kinase RNA-like endoplasmic reticulum kinase (PERK) ( G ), Eukaryotic translation initiation factor 2A (eIF2α) ( H ), and C/EBP Homologous Protein (CHOP) ( I ). Values represent the fold change relative to the control cells and are expressed as mean± standard error of mean, n = 3. * denotes p < 0.05 compared to control cells, # denotes p < 0.05 compared with the cells treated with gAcrp but not transfected with siRNA.

Article Snippet: Recombinant human globular adiponectin (gAcrp) was purchased from PeproTech Inc. (Rocky Hill, NJ, USA).

Techniques: Phospho-proteomics, Inhibition, Expressing, Western Blot, Transfection, Incubation, Immunoprecipitation, Control

Inhibition of the inflammasome activation mediates modulation of breast cancer cell growth. ( A – D ) MCF-7 cells were treated with pharmacological inhibitors of inflammasomes ( A , C ), including Ac-YVAD-cmk (10 µM), a caspase-1 inhibitor, MCC950 (10 µM), a small molecule inhibitor of NLRP3, or interleukin-1 receptor antagonist (1 µg/mL), or gAcrp (1 µg/mL) ( B , D ) for the indicated time duration. Cell viability ( A , B ) and Caspase-7 enzyme activity ( C , D ) were then determined as described in the Materials and Methods. ( E ) MCF-7 cells were incubated with gAcrp (1 µg/mL) or pharmacological inhibitors of inflammasomes for 48 h followed by staining with propidium iodide and flow cytometric analysis. ( F – O ) MCF-7 cells were incubated with gAcrp (1 µg/mL) ( F – J ) or pharmacological inhibitors of inflammasomes ( L – O ) for the indicated time periods. Immunoblot analysis was performed to determine the expression levels of Bcl2-associated X protein (Bax) ( F , K ), B-cell lymphoma 2 (Bcl-2) ( G , L ), cyclin D1 ( H , M ), p27 ( I , N ), and p53 ( J , O ). Images are the representative for three independent experiments that showed similar results. Values represent the fold change relative to the control cells and are expressed as mean± standard error of mean, n =3. * denotes p < 0.05 compared to control cells.

Journal: Cancers

Article Title: Globular Adiponectin Inhibits Breast Cancer Cell Growth through Modulation of Inflammasome Activation: Critical Role of Sestrin2 and AMPK Signaling

doi: 10.3390/cancers12030613

Figure Lengend Snippet: Inhibition of the inflammasome activation mediates modulation of breast cancer cell growth. ( A – D ) MCF-7 cells were treated with pharmacological inhibitors of inflammasomes ( A , C ), including Ac-YVAD-cmk (10 µM), a caspase-1 inhibitor, MCC950 (10 µM), a small molecule inhibitor of NLRP3, or interleukin-1 receptor antagonist (1 µg/mL), or gAcrp (1 µg/mL) ( B , D ) for the indicated time duration. Cell viability ( A , B ) and Caspase-7 enzyme activity ( C , D ) were then determined as described in the Materials and Methods. ( E ) MCF-7 cells were incubated with gAcrp (1 µg/mL) or pharmacological inhibitors of inflammasomes for 48 h followed by staining with propidium iodide and flow cytometric analysis. ( F – O ) MCF-7 cells were incubated with gAcrp (1 µg/mL) ( F – J ) or pharmacological inhibitors of inflammasomes ( L – O ) for the indicated time periods. Immunoblot analysis was performed to determine the expression levels of Bcl2-associated X protein (Bax) ( F , K ), B-cell lymphoma 2 (Bcl-2) ( G , L ), cyclin D1 ( H , M ), p27 ( I , N ), and p53 ( J , O ). Images are the representative for three independent experiments that showed similar results. Values represent the fold change relative to the control cells and are expressed as mean± standard error of mean, n =3. * denotes p < 0.05 compared to control cells.

Article Snippet: Recombinant human globular adiponectin (gAcrp) was purchased from PeproTech Inc. (Rocky Hill, NJ, USA).

Techniques: Inhibition, Activation Assay, Activity Assay, Incubation, Staining, Western Blot, Expressing, Control

Suppressive effects of globular adiponectin on NLRP3 inflammasomes activation in MCF-7 tumor xenograft model and its potential role in the modulation of growth of breast cancer cells. After xenograft tumors were generated in BALB/c nude male mice, the animals were randomly divided into four groups of 5 mice and intratumorally administered with gAcrp (2µg/mouse), MCC950 (40µg/mouse), IL-1Ra (2µg/mouse) or phosphate-buffered saline (control group) every 48 h for 3 weeks. ( A ) Representative images of mice bearing xenografted tumors from each group at the end of the treatment period. ( B ) Tumor tissues were isolated from each mouse after 3 weeks of treatment. ( C ) Tumor growth rate were monitored every three days during treatment. ( D ) After 3 weeks treatment, tumor tissues from each mouse were collected and weighed. ( E ) Tissue sections were prepared from tumor of each mouse and subjected to immunohistochemistry (IHC) staining for Ki-67. Scale bar: 5µm. ( F – H ) The expression levels of the proliferative and apoptotic markers, such as cyclin D1, p27, B-cell lymphoma 2 (Bcl-2), and Bcl2-associated X protein (Bax) ( F), and genes related to inflammasomes, including IL-1β and caspase-1 ( G), NLRP3, and the apoptosis-associated speck-like protein containing a CARD (ASC) ( H), were determined by Western blot. ( I ) The expression levels of NLRP3 and ASC were further confirmed by IHC. For IHC experiments, five mice were used in each group and statistical analysis was made with 3 different tumor sections of each animal. The percentage of staining positive cells (nuclear staining) or staining positive area (cytoplasmic staining) was determined using Image J Software version 1.52. Magnification 20×. Scale bar: 5µm. ( J ) Expression levels of AMP-activated protein kinase (AMPK), sestrin2 (SESN2), and C/EBP homologous protein (CHOP) were examined by Western blot analysis. For Western blot analyses, 5 mice were used in each group and the results from each mouse were included in the statistical analysis. Of the five samples, representative images for three samples in each group were presented. Values are expressed as mean± standard error of mean, n = 5. * denotes p < 0.05 compared with control group.

Journal: Cancers

Article Title: Globular Adiponectin Inhibits Breast Cancer Cell Growth through Modulation of Inflammasome Activation: Critical Role of Sestrin2 and AMPK Signaling

doi: 10.3390/cancers12030613

Figure Lengend Snippet: Suppressive effects of globular adiponectin on NLRP3 inflammasomes activation in MCF-7 tumor xenograft model and its potential role in the modulation of growth of breast cancer cells. After xenograft tumors were generated in BALB/c nude male mice, the animals were randomly divided into four groups of 5 mice and intratumorally administered with gAcrp (2µg/mouse), MCC950 (40µg/mouse), IL-1Ra (2µg/mouse) or phosphate-buffered saline (control group) every 48 h for 3 weeks. ( A ) Representative images of mice bearing xenografted tumors from each group at the end of the treatment period. ( B ) Tumor tissues were isolated from each mouse after 3 weeks of treatment. ( C ) Tumor growth rate were monitored every three days during treatment. ( D ) After 3 weeks treatment, tumor tissues from each mouse were collected and weighed. ( E ) Tissue sections were prepared from tumor of each mouse and subjected to immunohistochemistry (IHC) staining for Ki-67. Scale bar: 5µm. ( F – H ) The expression levels of the proliferative and apoptotic markers, such as cyclin D1, p27, B-cell lymphoma 2 (Bcl-2), and Bcl2-associated X protein (Bax) ( F), and genes related to inflammasomes, including IL-1β and caspase-1 ( G), NLRP3, and the apoptosis-associated speck-like protein containing a CARD (ASC) ( H), were determined by Western blot. ( I ) The expression levels of NLRP3 and ASC were further confirmed by IHC. For IHC experiments, five mice were used in each group and statistical analysis was made with 3 different tumor sections of each animal. The percentage of staining positive cells (nuclear staining) or staining positive area (cytoplasmic staining) was determined using Image J Software version 1.52. Magnification 20×. Scale bar: 5µm. ( J ) Expression levels of AMP-activated protein kinase (AMPK), sestrin2 (SESN2), and C/EBP homologous protein (CHOP) were examined by Western blot analysis. For Western blot analyses, 5 mice were used in each group and the results from each mouse were included in the statistical analysis. Of the five samples, representative images for three samples in each group were presented. Values are expressed as mean± standard error of mean, n = 5. * denotes p < 0.05 compared with control group.

Article Snippet: Recombinant human globular adiponectin (gAcrp) was purchased from PeproTech Inc. (Rocky Hill, NJ, USA).

Techniques: Activation Assay, Generated, Saline, Control, Isolation, Immunohistochemistry, Expressing, Western Blot, Staining, Software

Proposed model for the modulation of NOD-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasomes activation by globular adiponectin in breast cancer cells and its role in the suppression of tumor growth. Inflammasomes modulate tumor growth by complicated manners and its physiological roles would be depending on specific context. Herein, we clearly demonstrated that inflammasomes activation contributes to growth of breast cancer cells via activation of cell cycle and suppression of apoptosis. The physiological actions of adiponectin are initiated by binding with its specific transmembrane receptor (adiponectin receptor type 1 and type 2 (adipoR1 and adipoR2)). Binding of globular adiponectin with adipoR1/R2 generates signaling, which suppresses NLRP3 inflammasomes activation leading to the prevention of interleukin-1β (IL-1β) maturation and caspase-1 activation in both in vitro and in vivo xenograft models, implying that suppression of tumor growth by globular adiponectin is mediated via modulation of the inflammasome. Modulation of the inflammasome by globular adiponectin is mediated by sestrin2 (SESN2)/AMP-activated protein kinase (AMPK)/Endoplasmic reticulum (ER) stress axis. SESN2 induction leads to AMPK phosphorylation by promoting association of AMPK with its upstream kinase, LKB-1. Activation of AMPK signaling plays a pivotal role in reduction in ER stress, which subsequently leads to prevention of inflammasomes activation. In here, inflammasomes activation and IL-1β signaling might be required for survival and growth of breast tumor growth. Therefore, negative modulation of inflammasomes activation by globular adiponectin, MCC950, Ac-YVAD, and IL-1 receptor antagonist promotes apoptosis and impedes cancer cell proliferation. Therefore, modulation of inflammasomes and various molecules involved would be potential therapeutic targets for the treatment of breast cancer. The molecular mechanisms underlying SESN2 induction by globular adiponectin remain elusive. In addition, the molecular mechanisms by which ER stress induces inflammasomes activation and IL-1 signaling modulates apoptosis and cell cycle remain to be elucidated.

Journal: Cancers

Article Title: Globular Adiponectin Inhibits Breast Cancer Cell Growth through Modulation of Inflammasome Activation: Critical Role of Sestrin2 and AMPK Signaling

doi: 10.3390/cancers12030613

Figure Lengend Snippet: Proposed model for the modulation of NOD-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasomes activation by globular adiponectin in breast cancer cells and its role in the suppression of tumor growth. Inflammasomes modulate tumor growth by complicated manners and its physiological roles would be depending on specific context. Herein, we clearly demonstrated that inflammasomes activation contributes to growth of breast cancer cells via activation of cell cycle and suppression of apoptosis. The physiological actions of adiponectin are initiated by binding with its specific transmembrane receptor (adiponectin receptor type 1 and type 2 (adipoR1 and adipoR2)). Binding of globular adiponectin with adipoR1/R2 generates signaling, which suppresses NLRP3 inflammasomes activation leading to the prevention of interleukin-1β (IL-1β) maturation and caspase-1 activation in both in vitro and in vivo xenograft models, implying that suppression of tumor growth by globular adiponectin is mediated via modulation of the inflammasome. Modulation of the inflammasome by globular adiponectin is mediated by sestrin2 (SESN2)/AMP-activated protein kinase (AMPK)/Endoplasmic reticulum (ER) stress axis. SESN2 induction leads to AMPK phosphorylation by promoting association of AMPK with its upstream kinase, LKB-1. Activation of AMPK signaling plays a pivotal role in reduction in ER stress, which subsequently leads to prevention of inflammasomes activation. In here, inflammasomes activation and IL-1β signaling might be required for survival and growth of breast tumor growth. Therefore, negative modulation of inflammasomes activation by globular adiponectin, MCC950, Ac-YVAD, and IL-1 receptor antagonist promotes apoptosis and impedes cancer cell proliferation. Therefore, modulation of inflammasomes and various molecules involved would be potential therapeutic targets for the treatment of breast cancer. The molecular mechanisms underlying SESN2 induction by globular adiponectin remain elusive. In addition, the molecular mechanisms by which ER stress induces inflammasomes activation and IL-1 signaling modulates apoptosis and cell cycle remain to be elucidated.

Article Snippet: Recombinant human globular adiponectin (gAcrp) was purchased from PeproTech Inc. (Rocky Hill, NJ, USA).

Techniques: Activation Assay, Binding Assay, In Vitro, In Vivo, Phospho-proteomics, Biomarker Discovery

Effects of globular adiponectin on maturation and secretion of IL-1β stimulated by lipopolysaccharide (LPS) in murine peritoneal macrophages. ( A ) Macrophages were isolated from murine peritonea, pretreated with the indicated concentrations of gAcrp for 18 h, and stimulated with LPS (100 ng/mL) for 8 h and then ATP (5 mM) for 1 h. Total cellular lysates were prepared, and the levels of pro- and mature active IL-1β were determined by Western blot analysis. Representative images from three independent experiments are shown along with β-actin as an internal loading control; ( B ) after treatment with gAcrp, LPS, and ATP, as in A, media were collected, and the levels of mature active IL-1β were measured by Western blot analysis. Quantitative analysis of active IL-1β expression was performed by densitometric analysis and shown in the lower panel ( A , B ). Values presented are fold change compared to the cells treated with LPS and ATP and expressed as mean ± SEM ( n = 3 for A , n = 2 for B ). * p < 0.05 compared to the control cells. # p < 0.05 compared to the cells treated with LPS and ATP; ( C ) primary macrophages were isolated from murine peritonea, treated with gAcrp for 18 h, and stimulated with LPS (100 ng/mL) for 24 h and ATP for 1 h. The amount of secreted IL-1β was determined by ELISA as described in materials and methods. Results are presented as the mean ± SEM, n = 3. * p < 0.05 compared to control cells; # p < 0.05 compared to cells treated with LPS and ATP; ( D ) peritoneal macrophages were pretreated with gAcrp for 18 h, followed by stimulation with LPS (100 ng/mL) for 8 h and ATP for 1 h. IL-1β mRNA levels were determined by quantitative RT-PCR as indicated in materials and methods and normalized to the levels of GAPDH mRNA. Values indicate the fold increase compared to cells treated with LPS plus ATP and are expressed as the mean ± SEM ( n = 4). * p < 0.05 compared with untreated cells; # p < 0.05 compared to cells treated with LPS and ATP.

Journal: International Journal of Molecular Sciences

Article Title: Globular Adiponectin Inhibits Lipopolysaccharide-Primed Inflammasomes Activation in Macrophages via Autophagy Induction: The Critical Role of AMPK Signaling

doi: 10.3390/ijms18061275

Figure Lengend Snippet: Effects of globular adiponectin on maturation and secretion of IL-1β stimulated by lipopolysaccharide (LPS) in murine peritoneal macrophages. ( A ) Macrophages were isolated from murine peritonea, pretreated with the indicated concentrations of gAcrp for 18 h, and stimulated with LPS (100 ng/mL) for 8 h and then ATP (5 mM) for 1 h. Total cellular lysates were prepared, and the levels of pro- and mature active IL-1β were determined by Western blot analysis. Representative images from three independent experiments are shown along with β-actin as an internal loading control; ( B ) after treatment with gAcrp, LPS, and ATP, as in A, media were collected, and the levels of mature active IL-1β were measured by Western blot analysis. Quantitative analysis of active IL-1β expression was performed by densitometric analysis and shown in the lower panel ( A , B ). Values presented are fold change compared to the cells treated with LPS and ATP and expressed as mean ± SEM ( n = 3 for A , n = 2 for B ). * p < 0.05 compared to the control cells. # p < 0.05 compared to the cells treated with LPS and ATP; ( C ) primary macrophages were isolated from murine peritonea, treated with gAcrp for 18 h, and stimulated with LPS (100 ng/mL) for 24 h and ATP for 1 h. The amount of secreted IL-1β was determined by ELISA as described in materials and methods. Results are presented as the mean ± SEM, n = 3. * p < 0.05 compared to control cells; # p < 0.05 compared to cells treated with LPS and ATP; ( D ) peritoneal macrophages were pretreated with gAcrp for 18 h, followed by stimulation with LPS (100 ng/mL) for 8 h and ATP for 1 h. IL-1β mRNA levels were determined by quantitative RT-PCR as indicated in materials and methods and normalized to the levels of GAPDH mRNA. Values indicate the fold increase compared to cells treated with LPS plus ATP and are expressed as the mean ± SEM ( n = 4). * p < 0.05 compared with untreated cells; # p < 0.05 compared to cells treated with LPS and ATP.

Article Snippet: Recombinant human globular adiponectin (gAcrp) was obtained from Peprotech Inc. (Rocky Hill, NJ, USA).

Techniques: Isolation, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

Effects of globular adiponectin on lipopolysaccharide (LPS)-primed caspase-1 activation in peritoneal macrophages . Peritoneal macrophages were pretreated with gAcrp (0.1 μg/mL) for 18 h, followed by stimulation with LPS (100 ng/mL) for 6 h and then treatment with ATP (5 mM) for 30 min. ( A ) Pro- and cleaved active forms of caspase-1 were detected by Western blot analysis in peritoneal macrophages. Representative images from three independent experiments are shown along with β-actin as an internal loading control. Quantitative analysis of caspase-1 p20 expression was performed by densitometric analysis and shown in the lower panel. Values presented are fold change compared to LPS and ATP treatment and represented as mean ± SEM ( n = 3). * p < 0.05 compared to the control cells. # p < 0.05 compared to the cells treated with LPS and ATP; ( B ) cell lysates were prepared and used for the measurement of caspase-1 enzyme activity. Data are expressed as fold changes relative to cells treated with LPS and ATP. Values are presented as the mean ± SEM, n = 4. * p < 0.05 compared to cells treated with ATP; # p < 0.05 compared to cells treated with LPS and ATP; ( C ) Caspase-11 expression was measured by Western blot analysis. Representative images are shown along with β-actin as an internal loading control. Quantitative analysis of caspase-11 expression was performed by densitometric analysis and shown in the lower panel. Values presented are fold change compared to LPS and ATP treatment and presented as mean ± SEM ( n = 2). * p < 0.05 compared to the control cells. # p < 0.05 compared to the cells treated with LPS and ATP; ( D ) Caspase-8 expression levels were determined by Western blot analysis. Representative images from three independent experiments are shown along with β-actin as an internal loading control.

Journal: International Journal of Molecular Sciences

Article Title: Globular Adiponectin Inhibits Lipopolysaccharide-Primed Inflammasomes Activation in Macrophages via Autophagy Induction: The Critical Role of AMPK Signaling

doi: 10.3390/ijms18061275

Figure Lengend Snippet: Effects of globular adiponectin on lipopolysaccharide (LPS)-primed caspase-1 activation in peritoneal macrophages . Peritoneal macrophages were pretreated with gAcrp (0.1 μg/mL) for 18 h, followed by stimulation with LPS (100 ng/mL) for 6 h and then treatment with ATP (5 mM) for 30 min. ( A ) Pro- and cleaved active forms of caspase-1 were detected by Western blot analysis in peritoneal macrophages. Representative images from three independent experiments are shown along with β-actin as an internal loading control. Quantitative analysis of caspase-1 p20 expression was performed by densitometric analysis and shown in the lower panel. Values presented are fold change compared to LPS and ATP treatment and represented as mean ± SEM ( n = 3). * p < 0.05 compared to the control cells. # p < 0.05 compared to the cells treated with LPS and ATP; ( B ) cell lysates were prepared and used for the measurement of caspase-1 enzyme activity. Data are expressed as fold changes relative to cells treated with LPS and ATP. Values are presented as the mean ± SEM, n = 4. * p < 0.05 compared to cells treated with ATP; # p < 0.05 compared to cells treated with LPS and ATP; ( C ) Caspase-11 expression was measured by Western blot analysis. Representative images are shown along with β-actin as an internal loading control. Quantitative analysis of caspase-11 expression was performed by densitometric analysis and shown in the lower panel. Values presented are fold change compared to LPS and ATP treatment and presented as mean ± SEM ( n = 2). * p < 0.05 compared to the control cells. # p < 0.05 compared to the cells treated with LPS and ATP; ( D ) Caspase-8 expression levels were determined by Western blot analysis. Representative images from three independent experiments are shown along with β-actin as an internal loading control.

Article Snippet: Recombinant human globular adiponectin (gAcrp) was obtained from Peprotech Inc. (Rocky Hill, NJ, USA).

Techniques: Activation Assay, Western Blot, Expressing, Activity Assay

Effects of globular adiponectin on the inflammasome activation and lactate dehydrogenase (LDH) release in murine peritoneal macrophages treated with lipopolysaccharide (LPS). Peritoneal macrophages were pretreated with gAcrp (0.1 μg/mL) for 18 h, stimulated with LPS (100 ng/mL) for 6 h, and then treated with ATP (5 mM) for 1 h. ( A ) ASC expression levels were determined by Western blot analysis. Representative images from three independent experiments are shown along with β-actin as an internal loading control; ( B ) NLRP3 expression levels were determined by Western blot analysis. Representative images from three independent experiments are shown along with β-actin as an internal loading control. Quantitative analyses for the measurement of ASC ( A ) and NLRP3 ( B ) expression were performed by densitometric analysis and shown in the lower panel. Values presented are fold change compared to LPS and ATP treatment and expressed as mean ± SEM ( n = 3). * p < 0.05 compared to the control cells. # p < 0.05 compared to the cells treated with LPS and ATP; ( C ) cells were stained with a specific antibody against ASC (green) and DAPI (blue). ASC speck formation was analyzed by fluorescent immunocytochemistry and is indicated by white arrows. Representative images from three independent experiments are presented. Quantitative analysis of ASC speck formation (dots) was performed by counting the cells that showing ASC-DAPI and shown in the lower panel. Values are expressed as a percentage of the cells that showing ASC-DAPI dots. * p < 0.05 compared to cells treated with ATP; # p < 0.05 compared to cells treated with LPS and ATP; ( D ) cell culture media were collected and used to measure LDH release. Values are shown as the mean ± SEM, n = 3. * p < 0.05 compared with cells treated with ATP; # p < 0.05 compared with cells treated with LPS and ATP.

Journal: International Journal of Molecular Sciences

Article Title: Globular Adiponectin Inhibits Lipopolysaccharide-Primed Inflammasomes Activation in Macrophages via Autophagy Induction: The Critical Role of AMPK Signaling

doi: 10.3390/ijms18061275

Figure Lengend Snippet: Effects of globular adiponectin on the inflammasome activation and lactate dehydrogenase (LDH) release in murine peritoneal macrophages treated with lipopolysaccharide (LPS). Peritoneal macrophages were pretreated with gAcrp (0.1 μg/mL) for 18 h, stimulated with LPS (100 ng/mL) for 6 h, and then treated with ATP (5 mM) for 1 h. ( A ) ASC expression levels were determined by Western blot analysis. Representative images from three independent experiments are shown along with β-actin as an internal loading control; ( B ) NLRP3 expression levels were determined by Western blot analysis. Representative images from three independent experiments are shown along with β-actin as an internal loading control. Quantitative analyses for the measurement of ASC ( A ) and NLRP3 ( B ) expression were performed by densitometric analysis and shown in the lower panel. Values presented are fold change compared to LPS and ATP treatment and expressed as mean ± SEM ( n = 3). * p < 0.05 compared to the control cells. # p < 0.05 compared to the cells treated with LPS and ATP; ( C ) cells were stained with a specific antibody against ASC (green) and DAPI (blue). ASC speck formation was analyzed by fluorescent immunocytochemistry and is indicated by white arrows. Representative images from three independent experiments are presented. Quantitative analysis of ASC speck formation (dots) was performed by counting the cells that showing ASC-DAPI and shown in the lower panel. Values are expressed as a percentage of the cells that showing ASC-DAPI dots. * p < 0.05 compared to cells treated with ATP; # p < 0.05 compared to cells treated with LPS and ATP; ( D ) cell culture media were collected and used to measure LDH release. Values are shown as the mean ± SEM, n = 3. * p < 0.05 compared with cells treated with ATP; # p < 0.05 compared with cells treated with LPS and ATP.

Article Snippet: Recombinant human globular adiponectin (gAcrp) was obtained from Peprotech Inc. (Rocky Hill, NJ, USA).

Techniques: Activation Assay, Expressing, Western Blot, Staining, Immunocytochemistry, Cell Culture

Role of autophagy induction in the suppression of IL-1β maturation and secretion by globular adiponectin in macrophages. ( A , B ) Macrophages were treated with gAcrp (0.1 μg/mL) for 18 h. ATG5 ( A ) and LC3 ( B ) expression levels were determined by Western blot analysis. Representative images from three independent experiments are shown along with β-actin as an internal loading control. ( C – E ) Cells were pretreated with gAcrp (0.1 μg/mL) for 18 h in the absence or presence of 3-MA or Bafilomycin, stimulated with LPS (100 ng/mL) for 8 h, and then treated with ATP (5 mM) for 1 h; ( C ) total cellular lysates were prepared, and the levels of pro- and mature active IL-1β were measured by Western blot analysis. Representative images from three independent experiments are shown along with β-actin as an internal loading control; ( D ) cell culture media were collected, and the levels of mature active IL-1β were measured by Western blot analysis. Quantitative analysis of active IL-1β (p17) was performed by densitometric analysis and shown in the lower panel. Values presented are fold change compared to LPS and ATP treatment and expressed as mean ± SEM ( n = 3). * p < 0.05 compared to the control cells. # p < 0.05 compared to the cells treated with LPS and ATP. $ p < 0.05 compared to the cells treated with gAcrp and LPS/ATP. (E) The amount of secreted IL-1β was determined by ELISA. Results are presented as the mean ± SEM, n = 3. * p < 0.05 compared with control cells; # p < 0.05 compared with the cells treated with LPS and ATP. $ p < 0.05 compared to the cells treated with gAcrp and LPS/ATP.

Journal: International Journal of Molecular Sciences

Article Title: Globular Adiponectin Inhibits Lipopolysaccharide-Primed Inflammasomes Activation in Macrophages via Autophagy Induction: The Critical Role of AMPK Signaling

doi: 10.3390/ijms18061275

Figure Lengend Snippet: Role of autophagy induction in the suppression of IL-1β maturation and secretion by globular adiponectin in macrophages. ( A , B ) Macrophages were treated with gAcrp (0.1 μg/mL) for 18 h. ATG5 ( A ) and LC3 ( B ) expression levels were determined by Western blot analysis. Representative images from three independent experiments are shown along with β-actin as an internal loading control. ( C – E ) Cells were pretreated with gAcrp (0.1 μg/mL) for 18 h in the absence or presence of 3-MA or Bafilomycin, stimulated with LPS (100 ng/mL) for 8 h, and then treated with ATP (5 mM) for 1 h; ( C ) total cellular lysates were prepared, and the levels of pro- and mature active IL-1β were measured by Western blot analysis. Representative images from three independent experiments are shown along with β-actin as an internal loading control; ( D ) cell culture media were collected, and the levels of mature active IL-1β were measured by Western blot analysis. Quantitative analysis of active IL-1β (p17) was performed by densitometric analysis and shown in the lower panel. Values presented are fold change compared to LPS and ATP treatment and expressed as mean ± SEM ( n = 3). * p < 0.05 compared to the control cells. # p < 0.05 compared to the cells treated with LPS and ATP. $ p < 0.05 compared to the cells treated with gAcrp and LPS/ATP. (E) The amount of secreted IL-1β was determined by ELISA. Results are presented as the mean ± SEM, n = 3. * p < 0.05 compared with control cells; # p < 0.05 compared with the cells treated with LPS and ATP. $ p < 0.05 compared to the cells treated with gAcrp and LPS/ATP.

Article Snippet: Recombinant human globular adiponectin (gAcrp) was obtained from Peprotech Inc. (Rocky Hill, NJ, USA).

Techniques: Expressing, Western Blot, Cell Culture, Enzyme-linked Immunosorbent Assay

Role of autophagy induction in the suppression of caspase-1 activation, inflammasomes activation, and pyroptosis by globular adiponectin. ( A – D ) Cells were pretreated with gAcrp (0.1 μg/mL) for 18 h in the absence or presence of 3-MA (5 mM), stimulated with LPS (100 ng/mL) for 6 h, and then treated with ATP (5 mM) for 30 min. * p < 0.05 compared to cells treated with ATP; # p < 0.05 compared to cells treated with LPS and ATP; $ p < 0.05 compared to cells treated with gAcrp and LPS/ATP. ( A ) Levels of pro- and cleaved forms of caspase-1 in murine peritoneal macrophages were measured by Western blot analysis. Quantitative analysis of cleaved caspase-1 (caspase-1 p20) expression was performed by densitometric analysis and shown in the lower panel. Values presented are fold change compared to LPS and ATP treatment and represented as mean ± SEM ( n = 3). * p < 0.05 compared to the control cells. # p < 0.05 compared to the cells treated with LPS and ATP. $ p < 0.05 compared to the cells treated with gAcrp and LPS/ATP; ( B ) the culture media from peritoneal macrophages were prepared and used in a caspase-1 enzyme activity assay. Data are expressed as fold changes relative to LPS and ATP treatment. Values are shown as the mean ± SEM, n = 5; ( C ) cells were stained with antibody specific for ASC (green) and DAPI (blue). ASC speck formation was analyzed by fluorescent immunocytochemistry. Representative images from three independent experiments are shown along with quantitation of ASC speck formation (dots) in the lower panel. Values are expressed as a percentage of the cells that showing ASC-DAPI dots. * p < 0.05 compared to cells treated with ATP; # p < 0.05 compared to cells treated with LPS and ATP; ( D ) cell culture media were prepared and used for measurement of the LDH released. Values are presented as the mean ± SEM, n = 4.

Journal: International Journal of Molecular Sciences

Article Title: Globular Adiponectin Inhibits Lipopolysaccharide-Primed Inflammasomes Activation in Macrophages via Autophagy Induction: The Critical Role of AMPK Signaling

doi: 10.3390/ijms18061275

Figure Lengend Snippet: Role of autophagy induction in the suppression of caspase-1 activation, inflammasomes activation, and pyroptosis by globular adiponectin. ( A – D ) Cells were pretreated with gAcrp (0.1 μg/mL) for 18 h in the absence or presence of 3-MA (5 mM), stimulated with LPS (100 ng/mL) for 6 h, and then treated with ATP (5 mM) for 30 min. * p < 0.05 compared to cells treated with ATP; # p < 0.05 compared to cells treated with LPS and ATP; $ p < 0.05 compared to cells treated with gAcrp and LPS/ATP. ( A ) Levels of pro- and cleaved forms of caspase-1 in murine peritoneal macrophages were measured by Western blot analysis. Quantitative analysis of cleaved caspase-1 (caspase-1 p20) expression was performed by densitometric analysis and shown in the lower panel. Values presented are fold change compared to LPS and ATP treatment and represented as mean ± SEM ( n = 3). * p < 0.05 compared to the control cells. # p < 0.05 compared to the cells treated with LPS and ATP. $ p < 0.05 compared to the cells treated with gAcrp and LPS/ATP; ( B ) the culture media from peritoneal macrophages were prepared and used in a caspase-1 enzyme activity assay. Data are expressed as fold changes relative to LPS and ATP treatment. Values are shown as the mean ± SEM, n = 5; ( C ) cells were stained with antibody specific for ASC (green) and DAPI (blue). ASC speck formation was analyzed by fluorescent immunocytochemistry. Representative images from three independent experiments are shown along with quantitation of ASC speck formation (dots) in the lower panel. Values are expressed as a percentage of the cells that showing ASC-DAPI dots. * p < 0.05 compared to cells treated with ATP; # p < 0.05 compared to cells treated with LPS and ATP; ( D ) cell culture media were prepared and used for measurement of the LDH released. Values are presented as the mean ± SEM, n = 4.

Article Snippet: Recombinant human globular adiponectin (gAcrp) was obtained from Peprotech Inc. (Rocky Hill, NJ, USA).

Techniques: Activation Assay, Western Blot, Expressing, Enzyme Activity Assay, Staining, Immunocytochemistry, Quantitation Assay, Cell Culture

Role of 5′AMP-activated protein kinase ( AMPK ) signaling in autophagy induction and suppression of IL-1β maturation by globular adiponectin. ( A ) Peritoneal macrophages were pretreated with gAcrp (0.1 μg/mL) for 24 h, followed by stimulation with LPS (100 ng/mL) for additional 30 min. Phosphorylation of AMPK was determined by Western blot analysis. Images are representative of three independent experiments showing similar results; ( B , C ) macrophages were treated with gAcrp (0.1 μg/mL) for 24 h in the absence or presence of compound C. ATG5 ( B ) and LC3 ( C ) expression levels were assessed by Western blot analysis. Representative images from three independent experiments are shown along with β-actin as an internal loading control; ( D ) cells were pretreated with gAcrp (0.1 μg/mL) for 24 h in the absence or presence of compound C, stimulated with LPS (100 ng/mL) for 6 h, and then treated with ATP (5 mM) for 30 min. Levels of pro- and mature active forms of IL-1β were determined by Western blot analysis. Representative images from three independent experiments are shown along with β-actin as an internal loading control.

Journal: International Journal of Molecular Sciences

Article Title: Globular Adiponectin Inhibits Lipopolysaccharide-Primed Inflammasomes Activation in Macrophages via Autophagy Induction: The Critical Role of AMPK Signaling

doi: 10.3390/ijms18061275

Figure Lengend Snippet: Role of 5′AMP-activated protein kinase ( AMPK ) signaling in autophagy induction and suppression of IL-1β maturation by globular adiponectin. ( A ) Peritoneal macrophages were pretreated with gAcrp (0.1 μg/mL) for 24 h, followed by stimulation with LPS (100 ng/mL) for additional 30 min. Phosphorylation of AMPK was determined by Western blot analysis. Images are representative of three independent experiments showing similar results; ( B , C ) macrophages were treated with gAcrp (0.1 μg/mL) for 24 h in the absence or presence of compound C. ATG5 ( B ) and LC3 ( C ) expression levels were assessed by Western blot analysis. Representative images from three independent experiments are shown along with β-actin as an internal loading control; ( D ) cells were pretreated with gAcrp (0.1 μg/mL) for 24 h in the absence or presence of compound C, stimulated with LPS (100 ng/mL) for 6 h, and then treated with ATP (5 mM) for 30 min. Levels of pro- and mature active forms of IL-1β were determined by Western blot analysis. Representative images from three independent experiments are shown along with β-actin as an internal loading control.

Article Snippet: Recombinant human globular adiponectin (gAcrp) was obtained from Peprotech Inc. (Rocky Hill, NJ, USA).

Techniques: Western Blot, Expressing

5′AMP-activated protein kinase (AMPK) signaling deficiency diminishes suppression of inflammasome activation by globular adiponectin by impairing autophagy induction. ( A ) Peritoneal macrophages and spleen cells were isolated from wild-type mice or macrophage-specific conditionally AMPK-deficient mice (AMPK −/− ). AMPK protein expression was determined by Western blot analysis; ( B ) peritoneal macrophages were isolated from wild-type mice or macrophage-specific conditionally AMPK-deficient mice. Cells were pretreated with the indicated concentrations of gAcrp for 18 h and then stimulated with LPS (100 ng/mL) for 8 h. LC3 protein levels were determined by Western blot analysis. Images are representative of three independent experiments showing similar results along with β-actin as an internal loading control. Quantitative analysis of LC3II / LC3I ratio was performed by densitometric analysis and shown in the lower panel. Values presented are fold change compared to LPS treatment and expressed as mean ± SEM ( n = 3). * p < 0.05 compared to the control cells. # p < 0.05 compared to the cells treated with LPS; ( C ) Caspase-1 enzymatic activity was assessed by caspase-1 colorimetric enzyme assay as indicated in materials and methods. Values are represented as the mean ± SEM, n = 3. * p < 0.05 compared with cells treated with ATP; # p < 0.05 compared with the cells treated with LPS and ATP; $ p < 0.05 compared with wild type mice-derived macrophages treated with ATP, LPS and gAcrp; ( D ) cell culture media were prepared and used to measure LDH release. Values are represented as the mean ± SEM, n = 6. * p < 0.05 compared to cells treated with ATP; # p < 0.05 compared to cells treated with LPS and ATP; $ p < 0.05 compared to wild-type mouse-derived macrophages treated with gAcrp, LPS, and ATP.

Journal: International Journal of Molecular Sciences

Article Title: Globular Adiponectin Inhibits Lipopolysaccharide-Primed Inflammasomes Activation in Macrophages via Autophagy Induction: The Critical Role of AMPK Signaling

doi: 10.3390/ijms18061275

Figure Lengend Snippet: 5′AMP-activated protein kinase (AMPK) signaling deficiency diminishes suppression of inflammasome activation by globular adiponectin by impairing autophagy induction. ( A ) Peritoneal macrophages and spleen cells were isolated from wild-type mice or macrophage-specific conditionally AMPK-deficient mice (AMPK −/− ). AMPK protein expression was determined by Western blot analysis; ( B ) peritoneal macrophages were isolated from wild-type mice or macrophage-specific conditionally AMPK-deficient mice. Cells were pretreated with the indicated concentrations of gAcrp for 18 h and then stimulated with LPS (100 ng/mL) for 8 h. LC3 protein levels were determined by Western blot analysis. Images are representative of three independent experiments showing similar results along with β-actin as an internal loading control. Quantitative analysis of LC3II / LC3I ratio was performed by densitometric analysis and shown in the lower panel. Values presented are fold change compared to LPS treatment and expressed as mean ± SEM ( n = 3). * p < 0.05 compared to the control cells. # p < 0.05 compared to the cells treated with LPS; ( C ) Caspase-1 enzymatic activity was assessed by caspase-1 colorimetric enzyme assay as indicated in materials and methods. Values are represented as the mean ± SEM, n = 3. * p < 0.05 compared with cells treated with ATP; # p < 0.05 compared with the cells treated with LPS and ATP; $ p < 0.05 compared with wild type mice-derived macrophages treated with ATP, LPS and gAcrp; ( D ) cell culture media were prepared and used to measure LDH release. Values are represented as the mean ± SEM, n = 6. * p < 0.05 compared to cells treated with ATP; # p < 0.05 compared to cells treated with LPS and ATP; $ p < 0.05 compared to wild-type mouse-derived macrophages treated with gAcrp, LPS, and ATP.

Article Snippet: Recombinant human globular adiponectin (gAcrp) was obtained from Peprotech Inc. (Rocky Hill, NJ, USA).

Techniques: Activation Assay, Isolation, Expressing, Western Blot, Activity Assay, Enzymatic Assay, Derivative Assay, Cell Culture

Proposed model for the suppression of lipopolysaccharide (LPS)-primed inflammasomes activation by globular adiponectin via autophagy induction in macrophages. Treatment with globular adiponectin induces activation of an autophagic process via 5′AMP-activated protein kinase ( AMPK ) signaling in murine macrophages. The detailed mechanisms underlying p62 induction by globular adiponectin in the context of AMPK signaling remain to be determined. LPS treatment has been shown to enhance expression of pro-IL-1β through TLR4 and NF-κB signaling. Extracellular ATP triggers the inflammasome activation through a series of biological processes leading to the cleavage and activation of pro-caspase-1, which further induces maturation and secretion of IL-1β and pyroptosis in immune cells. In this study, we clearly showed that globular adiponectin suppresses LPS-stimulated IL-1β maturation and pyroptosis via modulation of inflammasomes activation. Importantly, autophagy induction plays a crucial role in the modulation of inflammasomes activation by adiponectin. It is likely that adiponectin-induced autophagy activation regulates inflammasomes activation through inhibition of ASC speck formation and inflammasomes assembly. Furthermore, AMPK signaling plays a pivotal role in autophagy activation and prevention of inflammasomes activation by globular adiponectin. P2X7 receptor: Purinergic ATP receptor; PYD: PYRIN-PAAD-DAPIN domain; CARD: Caspase activation and recruitment domain.

Journal: International Journal of Molecular Sciences

Article Title: Globular Adiponectin Inhibits Lipopolysaccharide-Primed Inflammasomes Activation in Macrophages via Autophagy Induction: The Critical Role of AMPK Signaling

doi: 10.3390/ijms18061275

Figure Lengend Snippet: Proposed model for the suppression of lipopolysaccharide (LPS)-primed inflammasomes activation by globular adiponectin via autophagy induction in macrophages. Treatment with globular adiponectin induces activation of an autophagic process via 5′AMP-activated protein kinase ( AMPK ) signaling in murine macrophages. The detailed mechanisms underlying p62 induction by globular adiponectin in the context of AMPK signaling remain to be determined. LPS treatment has been shown to enhance expression of pro-IL-1β through TLR4 and NF-κB signaling. Extracellular ATP triggers the inflammasome activation through a series of biological processes leading to the cleavage and activation of pro-caspase-1, which further induces maturation and secretion of IL-1β and pyroptosis in immune cells. In this study, we clearly showed that globular adiponectin suppresses LPS-stimulated IL-1β maturation and pyroptosis via modulation of inflammasomes activation. Importantly, autophagy induction plays a crucial role in the modulation of inflammasomes activation by adiponectin. It is likely that adiponectin-induced autophagy activation regulates inflammasomes activation through inhibition of ASC speck formation and inflammasomes assembly. Furthermore, AMPK signaling plays a pivotal role in autophagy activation and prevention of inflammasomes activation by globular adiponectin. P2X7 receptor: Purinergic ATP receptor; PYD: PYRIN-PAAD-DAPIN domain; CARD: Caspase activation and recruitment domain.

Article Snippet: Recombinant human globular adiponectin (gAcrp) was obtained from Peprotech Inc. (Rocky Hill, NJ, USA).

Techniques: Activation Assay, Expressing, Inhibition

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