Description

The INF2 Knockout HeLa Cell Line is a CRISPR/Cas9-edited knockout cell line engineered for permanent disruption of the INF2 gene in a HeLa host background. This loss-of-function model provides a stable and reproducible tool for dissecting INF2-dependent mechanisms without the variability of transient silencing approaches. By abrogating INF2 expression, researchers can investigate how this actin nucleation factor governs mitochondrial morphology, ER?Cmitochondria communication, and cytoskeletal dynamics in an epithelial cell context. The cell line is supplied as a validated knockout population, ready for expansion and downstream applications in cell biology, signal transduction, and disease modeling.

HeLa cells, the host for this knockout line, are an immortalized epithelial cell line originally derived from cervical carcinoma tissue. Their robust proliferation, ease of transfection, and well-characterized signaling networks make HeLa cells a workhorse for mechanistic studies of actin regulation, organelle dynamics, and stress responses. Although non-podocyte, HeLa cells express the core machinery for INF2-mediated actin polymerization and mitochondrial fission, enabling relevant interrogation of INF2 function in a human epithelial setting. The genetic tractability of HeLa cells facilitates integration of this knockout line with complementary tools such as rescue constructs, fluorescent reporters, and pharmacological perturbations.

INF2 acts as a formin-family actin nucleation factor that promotes linear actin filament assembly at endoplasmic reticulum?Cmitochondria contact sites. There, INF2-dependent actin polymerization drives the recruitment and oligomerization of the dynamin-related GTPase Drp1, leading to mitochondrial outer membrane constriction and subsequent fission. INF2 activity is regulated by RhoA?CROCK signaling, calcium influx, CDC42, and the ER stress sensor PERK. Among its interacting partners are profilin, actin, myosin II, Drp1, PERK, calmodulin, and Spire1. Downstream of INF2-mediated fission, mitochondrial reactive oxygen species can activate the NF-??B pathway, linking INF2 to inflammatory signaling. Thus, INF2 sits at the nexus of cytoskeletal organization, mitochondrial quality control, and cellular stress adaptation.

In the HeLa model, loss of INF2 disrupts actin dynamics at ER?Cmitochondria interfaces, impairing Drp1 recruitment and mitochondrial fission. This leads to elongated mitochondrial networks, altered bioenergetics, and potential dysregulation of NF-??B signaling. Because INF2 mutations are associated with focal segmental glomerulosclerosis and Charcot-Marie-Tooth disease, this cell line enables investigation of fundamental cellular defects relevant to these conditions without requiring specialized primary podocyte or neuronal cultures. Additionally, INF2 knockout HeLa cells provide a platform to study the interplay between the actin cytoskeleton and mitochondrial homeostasis in an oncogenic epithelial background, offering insights into cancer cell metabolism and stress resilience.

This knockout cell line supports a wide range of assays for mitochondrial and cytoskeletal research. Immunofluorescence with MitoTracker or anti-Drp1 antibodies visualizes fission defects; Western blotting confirms loss of INF2 and assesses Drp1 and downstream effector levels; co-immunoprecipitation maps INF2?CDrp1 interaction changes. Functional studies can employ actin polymerization assays, RhoA activation kits, and cell migration or adhesion assays. The model is suitable for drug screening targeting mitochondrial dynamics or ER stress, as well as for mechanistic dissection of RhoA?CROCK?CINF2 signaling. For further information or custom solutions, please contact Ascent Research.