The ATG3 Knockout 786-O Polyclonal Cells constitute a CRISPR/Cas9-edited polyclonal knockout cell population derived from the human 786-O renal cell carcinoma line, engineered to disrupt the ATG3 gene. This polyclonal population offers a heterogeneous pool of cells with targeted gene inactivation, avoiding clonal selection biases and better recapitulating genetic heterogeneity. It provides a flexible loss-of-function model for investigating autophagy without the limitations of single-cell-derived clones.
The 786-O cell line is a VHL-mutated clear cell renal adenocarcinoma line established from a primary kidney tumor. Its VHL deficiency mimics the most common genetic alteration in ccRCC, leading to constitutive HIF activation and metabolic reprogramming. This background is highly relevant for studying the interplay between autophagy, tumor metabolism, and hypoxia-driven pathways.
ATG3 functions as an E2-like enzyme in the ubiquitin-like conjugation system that lipidates ATG8 family proteins (LC3B, GABARAP) on autophagosomal membranes. The reaction is dependent on ATG7 (E1) and the ATG12-ATG5-ATG16L1 complex (E3), and is regulated by ATG4-mediated processing. Upstream signaling includes mTORC1 inhibition and AMPK activation, which converge on the ULK1 complex and PI3KC3 complex I (BECN1, ATG14, PIK3R4, PIK3C3) to initiate autophagy. ATG3-mediated lipidation promotes autophagosome maturation and degradation of cargoes such as p62/SQSTM1, damaged mitochondria, and protein aggregates.
In 786-O cells, autophagy is often subverted to support tumor survival under stress. Disrupting ATG3 in this VHL-mutant environment enables dissection of autophagy??s role in renal carcinoma proliferation, adaptation to hypoxia, and resistance to therapies. The model allows exploration of crosstalk between the autophagic machinery and the VHL/HIF pathway, which may reveal therapeutic vulnerabilities.
Typical experimental approaches include LC3B/p62 Western blotting and immunofluorescence for autophagy flux analysis, with Bafilomycin A1 to block degradation. Interaction studies via co-immunoprecipitation can validate ATG3 association with ATG7 or LC3 proteins. Cell proliferation, wound healing, and drug resistance assays are suited to assess tumorigenic potential. RNA-sequencing can profile transcriptional changes in autophagy and related pathways. For additional technical details, contact Ascent Research.
