研究目的
Investigating the role of pannexin-1 in canonical and noncanonical inflammasome activation.
研究成果
Pannexin-1 is required for driving NLRP3 inflammasome assembly during apoptosis but is dispensable for canonical NLRP3 or noncanonical inflammasome activation in macrophages.
研究不足
The study cannot rule out the possibility that pannexin-1 may confer susceptibility to murine models of endotoxin shock in vivo.
1：Experimental Design and Method Selection:
Utilized three different pannexin-1 channel inhibitors and two lines of Panx1–/– macrophages to provide genetic and pharmacological evidence.
2：Sample Selection and Data Sources:
Bone marrow-derived macrophages (BMDMs) from WT and Panx1–/– mice.
3：List of Experimental Equipment and Materials:
Pannexin-1 channel inhibitors (probenecid, trovafloxacin, spironolactone), ABT-737, S63845, TNF, AZD5582, LPS, nigericin, ATP, MSU.
4：Experimental Procedures and Operational Workflow:
BMDMs were stimulated with various agents to induce apoptosis or inflammasome activation in the presence or absence of pannexin-1 inhibitors.
5：Data Analysis Methods:
Immunoblotting, LDH assay, IL-1β ELISA, statistical analyses using GraphPad Prism 7 software.
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ABT-737
Selleckchem
Induces intrinsic apoptosis
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S63845
Selleckchem
MCL1 inhibitor
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TNF
PeproTech
Induces extrinsic apoptosis
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AZD5582
Selleckchem
SMAC-mimetic
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probenecid
Sigma
Pannexin-1 channel inhibitor
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trovafloxacin
Sigma
Pannexin-1 channel inhibitor
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spironolactone
Sigma
Pannexin-1 channel inhibitor
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LPS
InvivoGen
Priming agent for inflammasome activation
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nigericin
InvivoGen
Canonical NLRP3 inflammasome agonist
-
ATP
Sigma
Canonical NLRP3 inflammasome agonist
-
MSU
InvivoGen
Particulate NLRP3 inflammasome agonist
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Pam3CSK4
InvivoGen
Priming agent for noncanonical inflammasome activation
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Fugene HD
Promega
Transfection reagent
-
DMEM
Gibco
Cell culture medium
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FCS
Bioconcept
Cell culture supplement
-
HEPES
Bioconcept
Cell culture supplement
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penicillin/streptomycin
Bioconcept
Cell culture supplement
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nonessential amino acids
Gibco
Cell culture supplement
-
Opti-MEM
Gibco
Cell culture medium
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TaKaRa LDH cytotoxicity detection kit
Clontech
LDH assay
-
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