Journal: International immunology
Article Title: The human NAIP-NLRC4-inflammasome senses the Pseudomonas aeruginosa T3SS inner-rod protein.
doi: 10.1093/intimm/dxx047
Figure Lengend Snippet: Fig. 4. hNAIP-NLRC4 sensing of PscI-elicited human macrophages and PBMCs. Human monocyte cell line THP-1 (A–D) was differentiated into macrophages with PMA, and PBMCs (E) were isolated from peripheral venous blood. (A, B) Cells were seeded in 96-well culture plates and treated with fresh medium or 500 ng per well of ligands PscI, PscF, PopB, PrgI and MSU or 107 freeze-dried HKLM per well. Prior to treat- ment, cells were either left untreated or treated with the NLRP3 inhibitor Bay11-7082. The cytotoxicity (A) and the secretion of IL-1β (B) were determined 24 h post-contact by LDH release and ELISA. THP-1 cells (C, D) or PBMCs (E) were seeded in 24-well culture plates and were then transfected with NAIP1, NLRC4 or control siRNA. PscI at 500 ng or control medium were added per well. Relative gene expression was determined, 2 h post-contact, by quantitative RT–PCR (C); cytotoxicity and IL-1β secretion were determined by LDH release and ELISA (D, E). *P < 0.05; **P < 0.01; ***P < 0.001 compared to control.
Article Snippet: Inhibitors were used following the manufacturer’s instructions. siRNA transfection For siRNAs knockdown, THP-1 cells were differentiated in 24-well plates (106 cells per well). siRNAs specific for NLRC4 and NAIP1 were transfected into macrophages using a siRNA Reagent System (sc-45064, Santa Cruz Biotechnology, Santa Cruz, CA, USA) according to the manufacturer’s instructions. siRNAs were purchased from Santa Cruz Biotechnology.
Techniques: Isolation, Enzyme-linked Immunosorbent Assay, Transfection, Control, Gene Expression, Quantitative RT-PCR
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