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11 protocols using mcc950

1

Inflammasome Activation and Inhibition Assays

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MCC950 was purchased from TargetMol (Wellesley Hills, MA). Phorbol myristate acetate (PMA), ammonium chloride (NH4Cl), chloroquine diphosphate (CQ), N-acetyl-cysteine (NAC), pyrrolidine dithiocarbamate (PDTC), potassium chloride (KCl), glybenclamide, probenecid, carbenoxolone and cyclosporine A were purchased from Sigma-Aldrich (St. Louis, MO). Manganese (III) tetrakis (4-benzoic acid) porphyrin chloride (MnTBAP) and antibodies against ASC, IL-18, TLR4, P2X7, and actin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). PD98059, SB203580, SP600125, and antibodies against phospho-extracellular signal-regulated kinases 1/2 (ERK1/2), phospho-c-Jun N-terminal kinases 1/2 (JNK1/2), phospho-p38 and human caspase-1 were obtained from Cell Signaling Technology (Beverly, MA). Antibodies against NLRP3 and mouse caspase-1 were purchased from Adipogen International (San Diego, CA). Antibodies against IL-1β were purchased from R&D Systems (Minneapolis, MN). 10panx was purchased from Tocris Bioscience (Bristol, UK). The pHrodo Green E. coli BioParticles Conjugate was purchased from Thermo Fisher Scientific (Waltham, MA).
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2

LG Epithelial Cell Isolation and Treatments

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Mice were sacrificed by cervical dislocation after deep isoflurane-induced anesthesia (4% for induction). Their LGs were cut into small fragments (1 mm3) and treated with MEM-α and 0.1% type 2 collagenase under shaking on a shaker at 50 rpm at 37°C for 30 minutes. After blowing and mixing, as well as centrifuging at 1000 rpm for 5 minutes, the precipitate was uniformly dissolved in Dulbecco's modified Eagle's medium containing 20% fetal bovine serum. The supernatant was discarded again, and the resulting precipitate was fully mixed with Dulbecco's modified Eagle's medium containing 20% fetal bovine serum.
We treated the primary LG epithelial cells with MCC950 (10 µM/mL; TargetMol) for 24 hours in order to inhibit the NLRP3 inflammasome. For antioxidation experiments, the primary LG epithelial cells were treated with an N-acetyl-L-cysteine (NAC) antioxidant (1 mM; Sigma, St. Louis, MO, USA) for 24 hours. Thereafter, the treated primary LG epithelial cells of passage 2 were collected for Oil Red O (ORO) staining, JC-1 staining, TUNEL staining, and Western blotting.
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3

NLRP3 Inflammasome Activation Assay

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MSU, nigericin, ATP, poly(deoxyadenylic-thymidylic) acid sodium salt (poly (dA:dT)), polyinosinic:polycytidylic acid (poly (I:C)), phorbol-12-myristate-13-acetate (PMA), dimethyl sulfoxide (DMSO), and ultrapure LPS were purchased from Sigma–Aldrich (Munich, Germany). Pam3CSK4 was obtained from InvivoGen (Toulouse, France). MCC950 and CDN were from TargetMol (Boston, MA, USA). Lfn-Flic was kindly provided by Dr. Tao Li from National Center of Biomedical Analysis. Anti-mouse CASPASE-1 (1:1000, AG-20B-0042) was bought from Adipogen (San Diego, CA, USA). Anti-human cleaved IL-1β (1:2000, 12242), anti-human CASPASE-1 (1:2000, 4199 S), anti-mouse IL-1β (1:1000, 12507), anti-NLRP3 (1:2000, 15101S) were obtained from Cell Signaling Technology (Boston, MA, USA). Anti-ASC (1:1000, sc-22514-R) was from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-GAPDH (1:2000, 60004-1-1g) was purchased from Proteintech (Chicago, IL, USA).
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4

CRISPR/Cas9-Mediated AQP5 Knockout Mice

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The CRISPR/Cas9 technology was used to produce AQP5−/− C57BL/6 mice by Cyagen Biosciences (Guangzhou, China). All animal experiments were approved by The Ethics Committee Medical College of Qingdao University (No.QDU-AEC-2022297) and were conducted in accordance with the ARVO Statement on the Use of Animals in Ophthalmic and Vision Research.
Mice were intraperitoneally injected with MCC950 (100 µL, 20 mg/kg, TargetMol, Boston, MA, USA) once a week for 8 times in total. Then the samples were taken 1 week after the last injection of MCC950 for testing.
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5

Inflammasome Activation Assay

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Nigericin, ATP, poly(dA:dT), poly(I:C), phorbol-12-myristate-13-acetate (PMA), dimethyl sulfoxide (DMSO) and ultrapure LPS were obtained from Sigma-Aldrich (Munich, Germany). Silicon dioxide (SiO2) and Pam3CSK4 were obtained from InvivoGen (Toulouse, France). MCC950, carnosol, and geldanamycin (GA) were obtained from TargetMol (Boston, MA, USA). MitoTracker and MitoSOX were manufactured by Invitrogen (Carlsbad, CA, USA). Salmonella was a gift from Dr. Tao Li of the National Center of Biomedical Analysis. Anti-mouse Caspase-1 (1:1000, AG-20B-0042) was from Adipogen (San Diego, USA). Anti-human cleaved IL-1β (1:2000, 12242), Anti-mouse α-SMA (1:1000, 19245s), anti-human Caspase-1(1:2000, 4199S), anti-mouse IL-1β (1:1000, 12507) and anti-NLRP3 (1:2000, 15101S) were from Cell Signaling Technology (Boston, USA). Anti-ASC (1:1000, sc-22514-R) was from Santa Cruz Biotechnology (Dallas, USA). Anti-DDDK tag (1:3000, 20543–1-AP), Anti-HSP90 (1:3000, 13171-1-AP) and Anti-GAPDH (1:2000, 60004-1-1G) were from Proteintech Group (Chicago, USA).
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6

Induction of Accelerated Systemic Lupus Nephritis in NZB/WF1 Mice

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To establish ASLN model mice, 8-week-old female NZB/WF1 mice (prior to autoantibody production) were given LPS (Sigma, MO, USA) (0.8 mg/kg body weight) twice a week by intraperitoneal (ip) injections.15 (link) One week after the first injection of LPS, seven mice each received either Cf-02 (10 mg/kg body weight) or vehicle (PEG 400) (Sigma) daily via an ip route. Another group of such NZB/WF1 mice were treated with MCC950, a NLRP3 inhibitor (TargetMol, MA, USA), at 10 mg/kg body weight or saline every 2 days via ip injections. All mice were sacrificed 5 weeks after induction of the disease. Another group of age-matched female NZB/WF1 mice were injected with saline and served as normal controls. All animal experiments were performed in compliance with the NIH Guidelines for the Care and Use of Laboratory Animals with approval of the Institutional Animal Care and Use Committee of The National Defense Medical Center, Taipei, Taiwan.
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7

Immune Activation Reagents Protocol

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Adenosine triphosphate (ATP), nigericin, SiO2, poly (deoxyadenylic-thymidylic) acid sodium salt (poly (dA:dT)), polyinosinic: polycytidylic acid (poly (I:C)), Pam3CSK4, dimethyl sulfoxide (DMSO) and LPS (Escherichia coli, 055: B5) were purchased from Sigma-Aldrich (Munich, Germany). Epimedin A (110623–72-8, purity 99.0%), Epimedin A1 (140147–77-9, purity 99.92%), epimedin B (110623–73-9, purity 99.39%), epimedin C (110642–44-9, purity 99.1%), icariin (489–32-7, purity 97.64%), icaritin (118525–40-9, purity 99%), Icariside I (56725–99-6, purity 99.47%) and anhydroicaritin (38226–86-7, purity 99.51%) were purchased from TargetMol. Salmonella was kindly provided by Dr. Tao Li from National Center of Biomedical Analysis. MCC950 was obtained from TargetMol (Boston, USA). Anti-mouse caspase-1(1:1000, AG-20B-0042) was purchased from Adipogen (San Diego, USA). anti-mouse IL-1β (1:1000, 12,507), and anti-NLRP3 (1:2000, 15101S) were obtained from Cell Signaling Technology (Boston, USA). Anti-ASC (1:1000, sc-22,514-R) was purchased from Santa Cruz Biotechnology (Dallas, USA). Anti-GAPDH (1:2000, 60,004–1-1 g) was purchased from Proteintech (Chicago, USA). Color Prestained Protein marker (20AB01) was purchased from GenStar (Beijing, China).
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8

NLRP3 Inflammasome Activation Assay

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Adenosine triphosphate (ATP), Nigericin, SiO2, poly (deoxyadenylic-thymidylic) acid sodium salt (poly (dA:dT)), polyinosinic: polycytidylic acid [poly (I:C)], Pam3CSK4, dimethyl sulfoxide (DMSO), and LPS (Escherichia coli, 055: B5) were purchased from Sigma-Aldrich (Munich, Germany). Epimedin A (110623–72–8, purity 99.0%), Epimedin A1 (140147–77–9, purity 99.92%), epimedin B (110623–73–9, purity 99.39%), epimedin C (110642–44–9, purity 99.1%), icariin (489–32–7, purity 97.64%), caritin (118525–40–9, purity 99%), and anhydroicaritin (38226–86–7, purity 99.51%) were purchased from TargetMol. Salmonella strains were kindly provided by Dr. Tao Li from the National Center of Biomedical Analysis. MCC950 was obtained from TargetMol (Boston, MA, United States). Anti-mouse caspase-1(1:1000, AG-20B-0042) was purchased from Adipogen (San Diego, CA, United States). Anti-mouse IL-1β (1:1000, 12,507) and anti-NLRP3 (1:2000, 15101S) antibodies were obtained from Cell Signaling Technology (Boston, MA, United States). Anti-ASC (1:1000, sc-22,514-R) was purchased from Santa Cruz Biotechnology (Dallas, TX, United States). Anti-GAPDH (1:2000, 60,004–1-1 g) was purchased from Proteintech (Chicago, IL, United States). The color prestained protein marker (20AB01) was purchased from GenStar (Beijing, China).
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9

Investigation of Inflammatory Pathways

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Adenosine triphosphate (ATP), nigericin, SiO 2 , poly(deoxyadenylic-thymidylic) acid sodium salt (poly (dA:dT)), polyinosinic: polycytidylic acid (poly (I:C)), Pam3CSK4, dimethyl sulfoxide (DMSO) and LPS (Escherichia coli, 055: B5) were purchased from Sigma-Aldrich (Munich, Germany). Epimedin A (110623-72-8, purity 99.0%), Epimedin A 1 (140147-77-9, purity 99.92%), epimedin B (110623-73-9, purity 99.39%), epimedin C (110642-44-9, purity 99.1%), icariin (489-32-7, purity 97.64%), icaritin (118525-40-9, purity 99%), Icariside I (56725-99-6, purity 99.47%) and anhydroicaritin (38226-86-7, purity 99.51%) were purchased from TargetMol. Salmonella was kindly provided by Dr. Tao Li from National Center of Biomedical Analysis. MCC950 was obtained from TargetMol (Boston, USA). Anti-mouse caspase-1(1:1000, AG-20B-0042) was purchased from Adipogen (San Diego, USA). anti-mouse IL-1β (1:1000, 12507), and anti-NLRP3 (1:2000, 15101S) were obtained from Cell Signaling Technology (Boston, USA).
Anti-ASC (1:1000, sc-22514-R) was purchased from Santa Cruz Biotechnology (Dallas, USA). Anti-GAPDH (1:2000, 60004-1-1g) was purchased from Proteintech (Chicago, USA). Color Prestained Protein marker (20AB01) was purchased from GenStar (Beijing, China).
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10

Investigation of Inflammatory Pathways

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Adenosine triphosphate (ATP), nigericin, SiO 2 , poly(deoxyadenylic-thymidylic) acid sodium salt (poly (dA:dT)), polyinosinic: polycytidylic acid (poly (I:C)), Pam3CSK4, dimethyl sulfoxide (DMSO) and LPS (Escherichia coli, 055: B5) were purchased from Sigma-Aldrich (Munich, Germany). Epimedin A (110623-72-8, purity 99.0%), Epimedin A 1 (140147-77-9, purity 99.92%), epimedin B (110623-73-9, purity 99.39%), epimedin C (110642-44-9, purity 99.1%), icariin (489-32-7, purity 97.64%), icaritin (118525-40-9, purity 99%), Icariside I (56725-99-6, purity 99.47%) and anhydroicaritin (38226-86-7, purity 99.51%) were purchased from TargetMol. Salmonella was kindly provided by Dr. Tao Li from National Center of Biomedical Analysis. MCC950 was obtained from TargetMol (Boston, USA). Anti-mouse caspase-1(1:1000, AG-20B-0042) was purchased from Adipogen (San Diego, USA). anti-mouse IL-1β (1:1000, 12507), and anti-NLRP3 (1:2000, 15101S) were obtained from Cell Signaling Technology (Boston, USA).
Anti-ASC (1:1000, sc-22514-R) was purchased from Santa Cruz Biotechnology (Dallas, USA). Anti-GAPDH (1:2000, 60004-1-1g) was purchased from Proteintech (Chicago, USA). Color Prestained Protein marker (20AB01) was purchased from GenStar (Beijing, China).
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