The KLHL15 Knockout HCT 116 Polyclonal Cells product provides a heterogeneous population of HCT 116 colorectal carcinoma cells with CRISPR/Cas9-mediated disruption of the KLHL15 gene. This polyclonal knockout pool contains a variety of loss-of-function mutations, offering a robust model for functional studies without clonal isolation. As a mixed population, these cells are well-suited for bulk assays and initial screens.
HCT 116 is a human colorectal carcinoma epithelial cell line characterized by microsatellite instability (MSI) due to MLH1 deficiency, a KRAS G13D activating mutation, and wild-type TP53. This genetic background recapitulates a subset of colorectal cancers with defective DNA repair and constitutive mitogenic signaling, making it a valuable model for studying targeted therapies and proteasome dependencies.
KLHL15 encodes a substrate adaptor for the Cullin3-RING E3 ubiquitin ligase complex, which includes Cullin3 as a scaffold and RBX1 as the RING protein. Through its Kelch domain, KLHL15 recruits targets such as the PP2A-B55?? subunit (PPP2R2A) and the ORAI1 store-operated calcium channel for ubiquitination and proteasomal degradation. KLHL15 activity is regulated by cell cycle cues and mitogenic signals, and its degradation of PPP2R2A during mitosis modulates PP2A phosphatase activity to control cell cycle progression, while turnover of ORAI1 governs calcium entry. Thus, KLHL15 serves as a critical node linking ubiquitin-mediated proteolysis to cell division and calcium homeostasis.
In HCT 116 cells, knockout of KLHL15 is expected to stabilize substrates such as PPP2R2A and ORAI1, leading to elevated protein levels. Accumulation of PPP2R2A may alter PP2A holoenzyme composition and disrupt mitotic timing, while increased ORAI1 could enhance store-operated calcium influx, affecting calcium-dependent signaling pathways. Given that HCT 116 cells exhibit sensitivity to proteasome inhibition, loss of this specific E3 adaptor may uncover vulnerabilities or altered drug responses relevant to colorectal cancer. This model therefore provides a platform to dissect KLHL15-dependent regulation in a microsatellite-unstable, KRAS-mutant tumor context.
This polyclonal knockout cell population is applicable to a variety of experimental approaches, including Western blotting for substrate stabilization, ubiquitination assays, co-immunoprecipitation of the Cullin3 complex, and flow cytometry for cell cycle analysis. Additional uses include calcium imaging to monitor store-operated calcium entry, proliferation and colony formation assays, and proteasome activity measurements. The cells can also be employed in drug sensitivity studies to evaluate proteasome inhibitors or other targeted agents in the absence of KLHL15 function. For further technical details, please contact Ascent Research.
