DNAJC6 Knockout NCI-H1975 Polyclonal Cells represent a CRISPR/Cas9-edited polyclonal knockout cell population derived from the NCI-H1975 human lung adenocarcinoma cell line. This polyclonal pool carries targeted disruption of the DNAJC6 gene, which encodes the J-domain protein auxilin, a critical co?chaperone in clathrin-mediated endocytosis. The cell population is generated by CRISPR/Cas9-mediated gene disruption, providing a heterogeneous knockout model suitable for studying loss-of-function phenotypes without clonal selection artifacts.
The NCI-H1975 cell line is a widely used model of non-small cell lung cancer (NSCLC) adenocarcinoma, established from a patient with an activating EGFR L858R mutation. This mutation drives constitutive signaling through MAPK/ERK and PI3K/AKT pathways, making cells highly dependent on EGFR for proliferation and survival. The line exhibits epithelial morphology and retains lung adenocarcinoma characteristics, providing a valuable substrate for investigating oncogenic signaling and endocytic trafficking of receptor tyrosine kinases.
DNAJC6 (auxilin) functions as a J-domain co?chaperone that recruits Hsc70/HSPA8 to clathrin?coated pits, where it interacts with clathrin heavy chain and the AP?2 complex to stimulate ATP?dependent uncoating of clathrin lattices. In the endocytic pathway, DNAJC6 operates downstream of AP?2 cargo selection and upstream of dynamin?mediated scission. Knockout impairs clathrin disassembly, disrupting the endocytic cycle and potentially affecting internalization of cargo receptors such as EGFR. Key components??clathrin, AP?2, dynamin, Hsc70, and endosomal sorting complexes??converge on vesicle budding and recycling processes sensitive to auxilin levels.
In the EGFR L858R mutant NCI-H1975 background, DNAJC6 knockout is expected to perturb clathrin-dependent endocytosis of EGFR itself, thereby altering receptor internalization kinetics and downstream signal attenuation. Because clathrin-mediated endocytosis plays a key role in regulating the duration and strength of oncogenic EGFR signaling, loss of auxilin may lead to sustained surface receptor levels and enhanced proliferative signaling, or, conversely, interfere with signaling compartmentalization. This polyclonal knockout model enables dissection of the interplay between the endocytic machinery and mutant EGFR-driven tumor cell biology, providing a unique tool for examining how trafficking defects contribute to cancer cell phenotypes.
Researchers can employ this knockout cell pool to investigate clathrin-mediated endocytosis using transferrin uptake, EGF internalization, immunofluorescence of clathrin-coated pits, and flow cytometry for surface receptors. The model suits probing DNAJC6??s role in EGFR trafficking and endocytosis-dependent drug delivery. Given the link of DNAJC6 mutations to juvenile-onset Parkinson??s disease, these cells serve as a paradigm for neurodegeneration-relevant endocytic defects. Applications include Western blotting for clathrin and EGFR, electron microscopy of endocytic structures, and siRNA rescue experiments. For further information, please contact Ascent Research.
