Knockout of Atg5 inhibits proliferation and promotes apoptosis of DF-1 cells

Authors: LIAO, ZHIHONG - South China Agricultural University; DAI, ZHENKAI - South China Agricultural University; CAI, CHENYU - South China Agricultural University; ZHANG, XINHENG - South China Agricultural University; LI, AIJUN - South China Agricultural University; Zhang, Huanmin; YAN, YIMING - South China Agricultural University; LIN, WECHENG - South China Agricultural University; WU, YU - South China Agricultural University; LI, HONGXIN - South China Agricultural University; LI, HAIYUN - South China Agricultural University; XIE, QINGMEI - South China Agricultural University

Submitted to: In Vitro Cellular and Developmental Biology - Animal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/13/2019
Publication Date: 4/25/2019
Citation: Liao, Z., Dai, Z., Cai, C., Zhang, X., Li, A., Zhang, H., Yan, Y., Lin, W., Wu, Y., Li, H., Li, H., Xie, Q. 2019. Knockout of Atg5 inhibits proliferation and promotes apoptosis of DF-1 cells. In Vitro Cellular and Developmental Biology - Animals. 55(5):341-348. https://doi.org/10.1007/s11626-019-00342-7.
DOI: https://doi.org/10.1007/s11626-019-00342-7

Interpretive Summary: An important gene known as ATG5 encoding an autophagy related protein has been functionally examined experimentally. Using the cutting-edge technology scientifically known as CRISPR/Cas9 assay, the ATG5 gene was removed from a sample of an avian cell line (DF-1). Through a series of bench-top experiment tests and comparison between the modified and the unmodified DF-1 cells, new knowledge has been gained on the biological functions of this ATG5 gene, which include the influence of the gene on cell proliferation and cell death (apoptosis). This finding represents a basic advancement on the function of a gene and an illustration of the instrumental importance of new technologies on genetics research.

Technical Abstract: Atg5, as a switch of cell autophagy and apoptosis, plays an important regulatory role in the occurrence and development of autophagy. Atg5 has been reported to involve the autophagy process but little in the apoptotic process. Here, we constructed an Atg5-/- DF-1 cell line using the CRISPR/Cas9 assay and confirmed the significant difference in growth kinetics between Atg5-/- DF-1 cells and wild-type DF-1 cells. Importantly, we found that Atg5 suppresses the cellular proliferation and induce the apoptosis in DF-1 cells by Hoechst’s staining, flow cytometry, and caspase activity assay. All these findings indicated that Atg5 plays an important role in the proliferation of DF-1 cells. On the other hand, we compared the expression of autophagy key proteins LC3 and P62 in Atg5 knockout cells and wild-type cells, and detected the aggregation point distribution of LC3 protein in cells by laser confocal technique; our results showed that Atg5 knockout inhibited autophagy compared with wild-type cells. The present findings further help to resolve the molecular mechanisms regulating Atg5 autophagy and apoptosis.