This product comprises a CRISPR/Cas9-edited polyclonal knockout cell population of A-549 cells bearing a targeted disruption of the ATPAF1 locus. The polyclonal nature provides a diverse pool of knockout genotypes, enabling robust assessment of ATPAF1 loss-of-function phenotypes without clonal selection artifacts. These cells serve as a versatile tool for investigating mitochondrial ATP synthase assembly and associated cellular processes.
The host A-549 cell line, derived from human lung adenocarcinoma, is a widely used alveolar type II-like epithelial model. A-549 cells harbor a KRAS G12S-activating mutation and retain wild-type p53, representing a non-small cell lung cancer (NSCLC) subtype with reliance on mitochondrial oxidative metabolism for survival and proliferation. This background is particularly relevant for studying metabolic vulnerabilities in KRAS-driven tumors.
ATPAF1 encodes a mitochondrial assembly factor required for F1-portion assembly of ATP synthase (Complex V). Mechanistically, ATPAF1 interacts with components ATP5A1, ATP5B, and ATP5O, and cooperates with ATPAF2 and TMEM70 to form functional F1 complexes. Its expression is regulated by transcriptional regulators PPARGC1A, NRF1, NFE2L2, TFAM, and HIF1A, linking it to mitochondrial biogenesis and stress responses. Downstream, ATPAF1 ablation disrupts ATP synthase activity, leading to reduced ATP production, decreased mitochondrial membrane potential, and impaired oxidative phosphorylation, prompting metabolic reprogramming.
In the context of KRAS-mutant A-549 cells, ATPAF1 knockout creates a state of mitochondrial Complex V deficiency, sensitizing the cells to energetic stress while activating compensatory pathways. This model allows researchers to dissect the interplay between oncogenic KRAS signaling and mitochondrial OXPHOS dependency, revealing potential synthetic lethal interactions. It also permits evaluation of how p53 wild-type status influences metabolic adaptation under ATP synthase insufficiency.
This polyclonal knockout product is suited for diverse functional assays, including real-time respirometry (Seahorse analysis), ATP level quantification, JC-1 mitochondrial membrane potential measurements, and western blotting of OXPHOS subunits. It enables investigation of mitochondrial encephalomyopathies and Leigh syndrome mechanisms, metabolic reprogramming in cancer, and drug sensitivity profiling with mitochondrial inhibitors like oligomycin. Additional applications comprise RT-qPCR of biogenesis genes, RNA-seq transcriptomics, and apoptosis assays. For further information or custom requirements, please contact Ascent Research.
