ITPRIP Knockout SK-HEP-1 Polyclonal Cells provide a versatile CRISPR/Cas9-edited polyclonal population derived from the SK-HEP-1 cell line, featuring targeted disruption of the inositol 1,4,5-trisphosphate receptor interacting protein (ITPRIP) gene. This loss-of-function model is designed for in-depth investigation of ITPRIP-mediated calcium sensitization and its downstream signaling networks in a human hepatic endothelial-like background. The polyclonal format ensures representation of diverse knockout alleles, enabling robust functional studies without the clonal biases inherent in monoclonal lines.
SK-HEP-1 is an immortalized cell line originally established from the ascites of a patient with adenocarcinoma of the liver. Despite its tumorigenic origin, SK-HEP-1 displays hallmark endothelial features, including expression of von Willebrand factor and uptake of acetylated low-density lipoprotein, making it a widely accepted model for liver sinusoidal endothelial cells (LSECs). This dual epithelial-endothelial phenotype underpins its use in studying hepatic tumor microenvironment interactions, endothelial-to-mesenchymal transition (EndMT), and liver cancer progression.
At the molecular level, ITPRIP functions as a sensitizer of ITPR1, ITPR2, and ITPR3 by stabilizing their interaction with IP3, thereby amplifying endoplasmic reticulum (ER) calcium release. Activated by upstream cues including Reelin, BDNF, and PKC-mediated phosphorylation, ITPRIP bridges extracellular signals to intracellular calcium oscillations. It directly binds DAB1 to transduce Reelin-VLDLR pathway signals, leading to calcium-dependent activation of CaMKII, calcineurin, and transcription factors NFAT and CREB. Independently, ITPRIP engages GABARAP and LC3 to promote autophagosome formation, linking calcium homeostasis to autophagy. Thus, ITPRIP knockout abrogates IP3R sensitization, diminishing IP3-evoked calcium transients and impairing both autophagic flux and apoptotic signaling.
Within SK-HEP-1 cells, ITPRIP disruption perturbs the calcium-dependent endothelial-like functions and tumorigenic behavior. Given its role in sensitizing IP3Rs, knockout cells exhibit blunted cytosolic calcium responses, which can attenuate CaMKII-NFAT and CREB transcriptional programs involved in proliferation, migration, and survival. This is particularly relevant in the hepatic cancer context, where Reelin-DAB1 signaling has been implicated in tumor invasiveness and EndMT. The model further enables dissection of autophagy??s role in liver cancer, as loss of ITPRIP reduces GABARAP-LC3-mediated autophagy, potentially sensitizing cells to metabolic stress or chemotherapeutic agents.
This polyclonal knockout product supports a broad array of experimental approaches: calcium imaging with Fluo-4 AM to quantify IP3-evoked calcium transients; co-immunoprecipitation to assess ITPRIP-ITPR interactions; RT-qPCR and Western blot analysis of DAB1, CaMKII, NFAT, and autophagy markers (LC3, GABARAP); flow cytometry for Annexin V/PI apoptosis assays; and transwell migration/invasion studies to evaluate EndMT. Additionally, LC3-GFP autophagy flux assays and immunofluorescence for IP3R localization provide deep phenotype characterization. These cells are well-suited for drug discovery efforts targeting IP3R function or the Reelin pathway, and for exploring the intersection of calcium signaling and autophagy in hepatocellular carcinoma. For technical inquiries, protocol recommendations, or bulk ordering, please contact Ascent Research.
