The DNAJC6 Knockout NCI-H1299 Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout cell population with disrupted DNAJC6 across a pooled NCI-H1299 population. This format provides a heterogeneous loss-of-function model, well suited for studying gene function in a tumor-relevant context. NCI-H1299 is a widely used human non-small cell lung carcinoma line derived from a lymph node metastasis, particularly valued for EGFR signaling and drug resistance studies. CRISPR/Cas9-mediated DNAJC6 disruption in this background enables dissection of auxilin function in endocytic trafficking and signal transduction.
Established from a lung adenocarcinoma lymph node metastasis, NCI-H1299 lacks activating EGFR kinase domain mutations, making it ideal for investigating wild-type EGFR signaling and resistance to targeted therapies. This adherent epithelial NSCLC line maintains active endocytic pathways and signal transduction networks critical for tumor progression and drug sensitivity. Its well-characterized genome and culture protocols facilitate generation of genetically modified derivatives such as this DNAJC6 polyclonal knockout population.
DNAJC6 encodes auxilin, a co-chaperone that specifically recruits Hsc70 (HSPA8) to clathrin-coated pits through its J-domain. Auxilin stimulates Hsc70 ATPase activity, catalyzing clathrin cage disassembly and facilitating endocytic vesicle uncoating and recycling. This function is essential for clathrin-mediated endocytosis, synaptic vesicle recycling, and the trafficking of receptors like EGFR. Auxilin directly interacts with clathrin heavy chain and the AP-2 adaptor complex, and operates in concert with dynamin, synaptojanin, and endophilin during vesicle scission and recycling. Thus, DNAJC6 integrates upstream signals from cellular stress, Hsp70 activation, and EGFR signaling to control clathrin dynamics and synaptic protein localization.
In NCI-H1299 cells, DNAJC6 knockout impairs clathrin-mediated endocytosis, potentially altering EGFR internalization and recycling. This may dysregulate downstream Akt signaling, which is frequently activated in NSCLC and contributes to survival and therapy resistance. Consequently, this polyclonal knockout model is valuable for investigating how endocytic defects influence EGFR inhibitor sensitivity and resistance. Additionally, given DNAJC6??s link to juvenile Parkinson disease, the model offers a non-neuronal platform to study endocytic dysfunction in neurodegeneration.
Researchers can employ western blotting to assess EGFR and Akt phosphorylation, immunofluorescence to visualize clathrin and EGFR co-localization, transferrin uptake and EGFR internalization assays to quantify endocytic capacity, and clathrin coat disassembly assays to measure auxilin-dependent uncoating. RT-qPCR confirms DNAJC6 disruption, while cell viability assays with EGFR inhibitors test drug response. This polyclonal knockout model is thus suited for mechanistic studies of endocytosis, EGFR trafficking, drug resistance in NSCLC, and neurological disease pathways. For technical inquiries or to explore custom applications, please contact Ascent Research.
