The RIPK2 Knockout A549 Cell Line provides a CRISPR/Cas9-edited, stable loss-of-function model for the RIPK2 gene in A549 human lung adenocarcinoma cells. This system eliminates RIPK2 protein expression, enabling definitive dissection of NOD1/2-mediated innate immune and inflammatory signaling without the variability of RNA interference. It serves as a clean genetic background for studies of RIPK2-dependent pathways in pulmonary infection and tumor biology.
The parental A549 cell line was derived from a 58-year-old Caucasian male with lung adenocarcinoma. These epithelial cells retain type II pneumocyte characteristics, including lamellar bodies and surfactant production, and form tight monolayers in vitro. A549 cells are a standard model for lung cancer biology, drug testing, and respiratory infection research because they express pattern-recognition receptors such as NOD1, NOD2, and various TLRs, making them responsive to a broad range of microbial stimuli.
RIPK2 is a serine/threonine kinase and essential adaptor for NOD1 and NOD2 innate immune receptors. Upon bacterial peptidoglycan sensing, NOD1/2 recruit RIPK2 through CARD-CARD interactions, triggering its oligomerization and K63-linked ubiquitination by XIAP and cIAP1/2. This activates the TAK1-TAB2/3 complex and IKK??/??, leading to I??B?? phosphorylation/degradation and NF-??B p50/p65 nuclear translocation. Concurrently, RIPK2 stimulates the MAPK cascade, including p38 and JNK, to activate AP-1. Target genes include pro-inflammatory cytokines (IL-6, IL-8, TNF??), chemokines (CXCL1, CXCL2), and antimicrobial defensins. RIPK2 also interacts with ATG16L1 to modulate autophagy. Upstream inputs include TNF?? and IL-1??, while negative regulators include A20 (TNFAIP3) and ERBIN. Additional interactors such as TRAF6, CARD9, and PELI3 contribute to RIPK2 signaling complexity.
In the A549 lung epithelium context, RIPK2 is central for innate immune responses to respiratory pathogens that activate NOD1/2, including Legionella pneumophila, Chlamydia pneumoniae, and Pseudomonas aeruginosa. Knockout of RIPK2 abrogates NOD1/2-dependent NF-??B and MAPK activation, creating a clean isogenic system for studying alternative signaling routes or rescuing with mutant RIPK2 alleles. Because RIPK2 influences autophagy and chemokine production, this line is also valuable for investigating inflammatory modulation within the lung tumor microenvironment, epithelial-to-mesenchymal transition, and metastatic behavior.
This knockout line supports diverse assays: MDP or peptidoglycan challenge followed by phospho-I??B??, phospho-p65, phospho-p38, and phospho-JNK immunoblotting; RT-qPCR and ELISA for IL-6, IL-8, and CXCL1; NF-??B luciferase reporter assays; co-immunoprecipitation to confirm loss of NOD2-RIPK2 interaction; immunofluorescence for RIPK2 filament formation; autophagy flux measurements via LC3 puncta; and cell viability, migration, and invasion studies. It is also suited for xenograft models of lung adenocarcinoma and for evaluating RIPK2 inhibitors. For further information, please contact Ascent Research.
