DNAJB14 Knockout Huh-7 Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout cell population offering a loss-of-function model for the DNAJB14 gene in Huh-7 hepatocellular carcinoma cells. This heterogeneous pool preserves genetic diversity, avoiding clonal artifacts, and is ideal for studying ER-associated degradation (ERAD) and the unfolded protein response (UPR). DNAJB14 disruption impairs retrotranslocation of misfolded ER proteins, leading to their accumulation. Supplied cryopreserved, these cells enable robust interrogation of proteostasis networks in a hepatic cancer context.
Huh-7 is a well-differentiated human hepatocellular carcinoma line with epithelial morphology, retaining key liver functions such as drug metabolism and hepatitis virus permissiveness. Its extensive use in liver biology and CRISPR editing makes it a fitting host for investigating ER stress, as hepatocytes rely heavily on secretory protein folding capacity. The DNAJB14 knockout in this background thus provides a physiologically relevant system to study ERAD disruption and its consequences in a cancer model.
DNAJB14 is a J-domain co-chaperone that recruits HSPA5/BiP and the VCP/p97 ATPase to extract misfolded ER proteins, facilitating their proteasomal degradation after ubiquitination by the HRD1 complex. Under ER stress triggered by tunicamycin or thapsigargin, UPR sensors PERK, IRE1, and ATF6 activate transcription factors XBP1, ATF4, and CHOP, which modulate ERAD and autophagy. DNAJB14 expression is regulated by HSF1 and ATF6. Loss of DNAJB14 disrupts this clearance pathway, causing accumulation of ERAD substrates and sustained UPR, with compensatory increases in autophagy markers like LC3.
In Huh-7 cells, ER stress influences tumor aggressiveness and drug sensitivity. The DNAJB14 knockout model allows dissection of how impaired ERAD affects hepatocellular carcinoma cell survival, proliferation, and therapeutic response. The liver’s high secretory burden renders it vulnerable to proteotoxicity, and the polyclonal nature of these cells captures tumor heterogeneity, enabling more clinically relevant studies of ER stress signaling and cross-talk with autophagy pathways potentially exploitable for therapeutic intervention.
Applications include Western blotting for BiP, CHOP, and LC3; RT-qPCR for XBP1 and ATF4 targets; and ERAD substrate degradation assays using CD3??-GFP. Tunicamycin sensitivity, proteasomal activity, and autophagy flux (p62/LC3) assays reveal functional consequences. Co-immunoprecipitation confirms lost DNAJB14-BiP/VCP interactions, and immunofluorescence visualizes ER stress. Flow cytometry quantifies apoptosis and cell cycle changes. For technical details or to integrate this model into your research, contact Ascent Research.
