Journal: Veterinary Research

Article Title: Staphylococcus aureus mediates pyroptosis in bovine mammary epithelial cell via activation of NLRP3 inflammasome

doi: 10.1186/s13567-022-01027-y

Figure Lengend Snippet: NLRP3 inflammasome regulates ASC speck formation during S. aureus . Fluorescence microscopy images of MAC-T cells immunoassayed for ASC (red) with or without MCC950 after 4 h of S. aureus treatment.

Article Snippet: Caspase-1 inhibitor VX765 and NLRP3 inflammasome inhibitor MCC950 were obtained from Glpbio (USA) and used at final concentrations of 100 and 15 μM.

Techniques: Fluorescence, Microscopy

Journal: Veterinary Research

Article Title: Staphylococcus aureus mediates pyroptosis in bovine mammary epithelial cell via activation of NLRP3 inflammasome

doi: 10.1186/s13567-022-01027-y

Figure Lengend Snippet: NLRP3 inflammasome activation by S. aureus is essential for the generation of GSDMD-N and the release of IL-1β and IL-18. A Activated caspase-1 and B GSDMD-N or released C IL-1β and D IL-18 of MAC-T cells treated with or without NLRP3 inhibitor MCC950 or caspase-1 inhibitor VX765 for 1.5 h prior to treatment with S. aureus for 4 h. E Levels of IL-1β and F IL-18 released after S. aureus treatment of MAC-T cells for 4 h in the presence or absence of 25, 50, or 75 mM KCl.

Article Snippet: Caspase-1 inhibitor VX765 and NLRP3 inflammasome inhibitor MCC950 were obtained from Glpbio (USA) and used at final concentrations of 100 and 15 μM.

Techniques: Activation Assay

Journal: Cell Proliferation

Article Title: Aconitine induces cardiomyocyte damage by mitigating BNIP3‐dependent mitophagy and the TNFα‐NLRP3 signalling axis

doi: 10.1111/cpr.12701

Figure Lengend Snippet: A‐B, The viability of H9c2 cells incubated with aconitine alone or a combination of aconitine and Z‐VAD, MCC950 or MCC950 plus Z‐VAD; C‐D, Western blot analysis of NLRP3 and RIPK pathway‐related proteins in total lysates of H9c2 cells after the indicated treatments (* P < .05)

Article Snippet: The NLRP3 inhibitor MCC950, TNFα inhibitor Enbrel and aconitine were obtained from Shanghai Selleck Chemical Co., Ltd. (Shanghai, China) and Meilun Biotech.

Techniques: Incubation, Western Blot

Journal: Cell Proliferation

Article Title: Aconitine induces cardiomyocyte damage by mitigating BNIP3‐dependent mitophagy and the TNFα‐NLRP3 signalling axis

doi: 10.1111/cpr.12701

Figure Lengend Snippet: A, Western blot analysis of NLRP3 pathway activation after aconitine incubation for 24 h with or without the NLRP3 inhibitor MCC950; B, The subcellular location of NLRP3 and ASC used to detect NLRP3 inflammasome formation after aconitine incubation for 24 h with or without MCC950, scale bar: 6 μm

Article Snippet: The NLRP3 inhibitor MCC950, TNFα inhibitor Enbrel and aconitine were obtained from Shanghai Selleck Chemical Co., Ltd. (Shanghai, China) and Meilun Biotech.

Techniques: Western Blot, Activation Assay, Incubation

Journal: Cell Proliferation

Article Title: Aconitine induces cardiomyocyte damage by mitigating BNIP3‐dependent mitophagy and the TNFα‐NLRP3 signalling axis

doi: 10.1111/cpr.12701

Figure Lengend Snippet: A, Western blot analysis of NLRP3 pathway activation after aconitine incubation for 24 h with or without the TNFα inhibitor Enbrel or BNIP3 shRNA. B, Immunofluorescence analysis of BNIP3 and TOM20 to detect mitophagy after aconitine incubation for 24 h with or without the TNFα inhibitor Enbrel, scale bar: 10 μm

Article Snippet: The NLRP3 inhibitor MCC950, TNFα inhibitor Enbrel and aconitine were obtained from Shanghai Selleck Chemical Co., Ltd. (Shanghai, China) and Meilun Biotech.

Techniques: Western Blot, Activation Assay, Incubation, shRNA, Immunofluorescence

Journal: Cell Proliferation

Article Title: Aconitine induces cardiomyocyte damage by mitigating BNIP3‐dependent mitophagy and the TNFα‐NLRP3 signalling axis

doi: 10.1111/cpr.12701

Figure Lengend Snippet: A, Masson's trichrome staining of myocardial tissue sections of control, aconitine treated and aconitine plus BNIP3‐OE rats, scale bar: 1 mm. B, immunofluorescent staining of TUNEL and TNFα in myocardial tissue sections of control, aconitine treated and aconitine plus BNIP3‐OE rats, scale bar: 1 mm. C, The immunohistochemistry analysis of NLRP3, LC3, CTnI and CaMKII in myocardial tissue sections of control, aconitine treated and aconitine plus BNIP3‐OE‐treated rats, scale bar: 50 μm. * P < .05 vs control. ** P < .01 vs control. ## P < .01 to aconitine‐treated group

Article Snippet: The NLRP3 inhibitor MCC950, TNFα inhibitor Enbrel and aconitine were obtained from Shanghai Selleck Chemical Co., Ltd. (Shanghai, China) and Meilun Biotech.

Techniques: Staining, Control, TUNEL Assay, Immunohistochemistry

Journal: Frontiers in Pharmacology

Article Title: Neuroprotective Effects of Qingnao Dripping Pills Against Cerebral Ischemia via Inhibiting NLRP3 Inflammasome Signaling Pathway: In Vivo and In Vitro

doi: 10.3389/fphar.2020.00065

Figure Lengend Snippet: Effect of QNDP on the expression of NLRP3, ASC, and cleaved caspase 1/pro-caspase 1 in MCAO rats. (A) Western blotting analysis of NLRP3, (B) Western blotting analysis of ASC, (C) Western blotting analysis of C-caspase 1/pro-caspase 1, (D) Immunofluorescent staining for NLRP3 (red), NeuN (green), the nuclei were stained blue with DAPI. Scale bar indicates 20 μm. Data are presented as the mean ± SEM, n = 3 per group, * p < 0.05, ** p < 0.01 compared with the sham group, ## p < 0.01 compared with the MCAO group.

Article Snippet: NLRP3 inflammasome inhibitor (MCC950) and 2% 2,3,5-triphenyltetrazolium chloride (TTC) solution were purchased from Sigma-Aldrich (USA).

Techniques: Expressing, Western Blot, Staining

Journal: Frontiers in Pharmacology

Article Title: Neuroprotective Effects of Qingnao Dripping Pills Against Cerebral Ischemia via Inhibiting NLRP3 Inflammasome Signaling Pathway: In Vivo and In Vitro

doi: 10.3389/fphar.2020.00065

Figure Lengend Snippet: Effect of QNDP on the expression of NLRP3, ASC, and cleaved caspase 1 following OGD in SH-SY5Y cells. (A) Western blotting bands of NLRP3, ASC, C-caspase 1, pro-caspase 1, (B) Cell survival rate using CCK-8 assay. Western blotting analysis of NLRP3 (C) , ASC (D) , C-caspase 1/pro-caspase 1 (E) . (F) Immunocytochemical staining for NLRP3 (red), the nuclei were stained blue with DAPI. Scale bar indicates 10 μm. Data are presented as the mean ± SEM, n = 3-5 batches of cells, ** p < 0.01 compared with the sham group, # p < 0.05, ## p < 0.01 compared with the MCAO group.

Article Snippet: NLRP3 inflammasome inhibitor (MCC950) and 2% 2,3,5-triphenyltetrazolium chloride (TTC) solution were purchased from Sigma-Aldrich (USA).

Techniques: Expressing, Western Blot, CCK-8 Assay, Staining

Journal: BMC Nephrology

Article Title: Adiponectin protects obesity-related glomerulopathy by inhibiting ROS/NF-κB/NLRP3 inflammation pathway

doi: 10.1186/s12882-021-02391-1

Figure Lengend Snippet: The sequences of primers for qRT-PCR

Article Snippet: MPC5 cells were transfected with siRNAs against adiponectin (100 nM) for 48 h using the Lipofectamine RNAiMAX Transfection Reagent (Invitrogen, CA, USA), incubated with an NLRP3 inhibitor MCC950 (100 nM, R&D, #5479/10), or treated with an NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC, 50 µM, Sigma, #P8765) for 24 h followed by sodium palmitate treatment.

Techniques:

Journal: BMC Nephrology

Article Title: Adiponectin protects obesity-related glomerulopathy by inhibiting ROS/NF-κB/NLRP3 inflammation pathway

doi: 10.1186/s12882-021-02391-1

Figure Lengend Snippet: PA induces the activation of NLRP3 inflammasome and oxidative stress in MPC5 cells. Cells were treated with 150µM PA for 24 h. a , Western Blot analysis of NLRP3, procaspase-1, cleaved caspase-1, and ASC. *** p < 0.001 vs. the control group. b , The mRNA levels of NLRP3 inflammasome-related proteins were detected by qRT-PCR analysis. ** p < 0.01, *** p < 0.001 vs. the control group. c , ELISA assays were performed to measure the contents of IL-18 and IL-1β in cell culture supernatants. *** p < 0.001 vs. the control group. d . ROS production was assessed using a fluorometry assay. *** p < 0.001 vs. the control group. Data are presented as mean ± SEM from three independent experiments

Article Snippet: MPC5 cells were transfected with siRNAs against adiponectin (100 nM) for 48 h using the Lipofectamine RNAiMAX Transfection Reagent (Invitrogen, CA, USA), incubated with an NLRP3 inhibitor MCC950 (100 nM, R&D, #5479/10), or treated with an NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC, 50 µM, Sigma, #P8765) for 24 h followed by sodium palmitate treatment.

Techniques: Activation Assay, Western Blot, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: BMC Nephrology

Article Title: Adiponectin protects obesity-related glomerulopathy by inhibiting ROS/NF-κB/NLRP3 inflammation pathway

doi: 10.1186/s12882-021-02391-1

Figure Lengend Snippet: Adiponectin attenuates PA-induced apoptosis, NLRP3 inflammasome activation, and podocyte injury in vitro. a , The viability of cells treated with adiponectin at different concentrations (1, 2, 4, 8 µg/ml) was evaluated by CCK-8 assay. ** p < 0.01 vs. the control group, # p < 0.05 vs. the PA group. b , The apoptosis rate was determined by flow cytometry. *** p < 0.001 vs. the control group, ### p < 0.001 vs. the PA group. c , ROS production was assessed by a fluorometric assay. *** p < 0.001 vs. the control group. d , qRT-PCR analysis of nephrin, podocin, desmin, NLRP3, caspase-1, and NF-κB. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. controls. e - f , Western Blot was performed to measure the protein levels of nephrin, podocin, desmin, NLRP3, pro-caspase1, cleaved caspase-1, ASC, NF-κB, and p-NF-κB. *** p < 0.001 vs. controls. g , The levels of IL-18 and IL-1β in cell culture supernatants were assessed by ELISA. *** p < 0.001 vs. controls. Data are presented as mean ± SEM from three independent experiments

Article Snippet: MPC5 cells were transfected with siRNAs against adiponectin (100 nM) for 48 h using the Lipofectamine RNAiMAX Transfection Reagent (Invitrogen, CA, USA), incubated with an NLRP3 inhibitor MCC950 (100 nM, R&D, #5479/10), or treated with an NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC, 50 µM, Sigma, #P8765) for 24 h followed by sodium palmitate treatment.

Techniques: Activation Assay, In Vitro, CCK-8 Assay, Flow Cytometry, Quantitative RT-PCR, Western Blot, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: BMC Nephrology

Article Title: Adiponectin protects obesity-related glomerulopathy by inhibiting ROS/NF-κB/NLRP3 inflammation pathway

doi: 10.1186/s12882-021-02391-1

Figure Lengend Snippet: The knockdown of adiponectin exacerbates intracellular inflammation and apoptosis in MPC5 cells. Podocytes were transfected with siRNA against adiponectin or NC siRNA. a , The transfection efficiency was evaluated by qRT-PCR and Western blot. *** p < 0.001 vs. the NC siRNA group. b , CCK-8 assay was used to evaluate cell viability after transfection. ** p < 0.01, *** p < 0.001 vs. controls. c , The percentage of apoptotic cells was determined after the transfection. * p < 0.05, ** p < 0.01,*** p < 0.001 vs. controls. d , The mRNA expressions of nephrin, podocin, desmin, NLRP3, caspase-1, and NF-κB were evaluated by qRT-PCR. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. controls. e - f , Western Blot analysis of nephrin, podocin, desmin, NLRP3, caspase-1, and NF-κB. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. controls. g , Levels of inflammatory cytokines IL-18 and IL-1β were assessed by ELISA. *** p < 0.001 vs. controls. Data are presented as mean ± SEM from three independent experiments

Article Snippet: MPC5 cells were transfected with siRNAs against adiponectin (100 nM) for 48 h using the Lipofectamine RNAiMAX Transfection Reagent (Invitrogen, CA, USA), incubated with an NLRP3 inhibitor MCC950 (100 nM, R&D, #5479/10), or treated with an NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC, 50 µM, Sigma, #P8765) for 24 h followed by sodium palmitate treatment.

Techniques: Transfection, Quantitative RT-PCR, Western Blot, CCK-8 Assay, Enzyme-linked Immunosorbent Assay

Journal: BMC Nephrology

Article Title: Adiponectin protects obesity-related glomerulopathy by inhibiting ROS/NF-κB/NLRP3 inflammation pathway

doi: 10.1186/s12882-021-02391-1

Figure Lengend Snippet: The NLRP3 inflammasome inhibitor MCC950 attenuated podocyte injury and intracellular oxidative stress induced by PA treatment or insufficient adiponectin expression. a , CCK-8 assay was performed to evaluate cell viability. * p < 0.05, *** p < 0.001 vs. controls. b , ROS production was assessed using a fluorometry assay. *** p < 0.001 vs. controls. c , The apoptosis was determined by flow cytometry. *** p < 0.001 vs. controls. d , qRT-PCR analysis of nephrin, podocin, desmin, NLRP3, caspase-1, and NF-κB. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. controls. e , Levels of inflammatory cytokines IL-1β and IL-18 were measured by ELISA. *** p < 0.001 vs. controls. f - g , Western blot analysis determined the protein levels of nephrin, podocin, desmin,NLRP3, procaspase-1, cleaved caspase-1, ASC, p65, and phosphorylated p65. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. controls. Data are presented as mean ± SEM from three independent experiments

Article Snippet: MPC5 cells were transfected with siRNAs against adiponectin (100 nM) for 48 h using the Lipofectamine RNAiMAX Transfection Reagent (Invitrogen, CA, USA), incubated with an NLRP3 inhibitor MCC950 (100 nM, R&D, #5479/10), or treated with an NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC, 50 µM, Sigma, #P8765) for 24 h followed by sodium palmitate treatment.

Techniques: Expressing, CCK-8 Assay, Flow Cytometry, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: BMC Nephrology

Article Title: Adiponectin protects obesity-related glomerulopathy by inhibiting ROS/NF-κB/NLRP3 inflammation pathway

doi: 10.1186/s12882-021-02391-1

Figure Lengend Snippet: Effects of PDTC on NLRP3 inflammasome activation and expression of podocyte makers in PA-stimulated podocytes. Podocytes subjected to adiponectin treatment (0 or 2 µg/mL) were preconditioned with or without 50 µM PDTC for 2 h, followed by exposure to 150 µM PA for 24 h. a , d The levels of NF-κB and p-NF-κB were detected by Western blot. b , e The levels of podocyte makers (nephrin, podocin, and desmin) were determined by Western blot. c , f The levels of NLRP3 inflammasome (NLRP3, cleaved caspase-1, procaspase-1, and ASC) were measured by Western blot. * p < 0.05, *** p < 0.001 vs. the control group. Data are presented as mean ± SEM from three independent experiments

Article Snippet: MPC5 cells were transfected with siRNAs against adiponectin (100 nM) for 48 h using the Lipofectamine RNAiMAX Transfection Reagent (Invitrogen, CA, USA), incubated with an NLRP3 inhibitor MCC950 (100 nM, R&D, #5479/10), or treated with an NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC, 50 µM, Sigma, #P8765) for 24 h followed by sodium palmitate treatment.

Techniques: Activation Assay, Expressing, Western Blot

Journal: BMC Nephrology

Article Title: Adiponectin protects obesity-related glomerulopathy by inhibiting ROS/NF-κB/NLRP3 inflammation pathway

doi: 10.1186/s12882-021-02391-1

Figure Lengend Snippet: Adiponectin alleviated ORG in obese mice. a , Kidney tissue sections show podocytes in both groups. b - c , The body weight and blood glucose level in all animals were measured at 6, 8, 10, and 12 weeks of age). *** p < 0.001 vs. CON group, # p < 0.05, ## p < 0.01 vs. HFD + gAd group. d , The serum concentrations of high-density lipoprotein, triglyceride, total cholesterol, and low-density lipoprotein were detected at 6, 8, 10, and 12 weeks of age. *** p < 0.001 vs. CON group, ### p < 0.001 vs. HFD + gAd group. e The levels of urinary ACR and serum were assessed at various time points (6, 8, 10, and 12 weeks of age). *** p < 0.001 vs. CON group, ### p < 0.001 vs. HFD + gAd group. f The level of serum adiponectin in mice was detected at 6 and 12 weeks of age. *** p < 0.001 vs. CON group. g - h Immunofluorescence stains of renal sections for podocin and NLRP3 at 12 weeks of age. * p < 0.05, *** p < 0.001 vs. CON group, ### p < 0.001 vs. HFD + gAd group. Data are presented as the mean ± SEM from three independent experiments

Article Snippet: MPC5 cells were transfected with siRNAs against adiponectin (100 nM) for 48 h using the Lipofectamine RNAiMAX Transfection Reagent (Invitrogen, CA, USA), incubated with an NLRP3 inhibitor MCC950 (100 nM, R&D, #5479/10), or treated with an NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC, 50 µM, Sigma, #P8765) for 24 h followed by sodium palmitate treatment.

Techniques: Immunofluorescence