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

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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.

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Funding

This study was supported by a grant from South China Agricultural University, the National Natural Science Foundation of China (Grant Nos. 31672564, 31472217), International Science and technology cooperation project of Guangdong Province (2016A050502042), Construction project of modern agricultural science and technology innovation alliance in Guangdong Province(2017LM1112), and the Natural Science Foundation of Guangdong Province (Grant No. S2013030013313).

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Authors and Affiliations

  1. College of Animal Science, South China Agricultural University and Guangdong Provincial Key Lab of Agro-Animal Genomics and Molecular Breeding and Key Laboratory of Chicken Genetics, Breeding and Reproduction, Ministry of Agriculture, Guangzhou, 510642, People’s Republic of China

    Zhihong Liao, Zhenkai Dai, Chenyu Cai, Xinheng Zhang, Yiming Yan, Wencheng Lin, Yu Wu, Hongxin Li, Haiyun Li & Qingmei Xie

  2. Key Laboratory of Animal Health Aquaculture and Environmental Control, Guangzhou, 510642, Guangdong, People’s Republic of China

    Zhihong Liao, Zhenkai Dai, Xinheng Zhang, Yiming Yan, Wencheng Lin, Yu Wu, Hongxin Li & Qingmei Xie

  3. Guangdong Provincial Animal Virus Vector Vaccine Engineering Technology Research Center, Guangzhou, 510642, People’s Republic of China

    Zhenkai Dai, Xinheng Zhang, Wencheng Lin, Hongxin Li & Qingmei Xie

  4. College of science and engineering, Jinan University, Guangzhou, 510632, People’s Republic of China

    Aijun Li

  5. Agriculture Research Service, Avian Disease and Oncology Laboratory, USDA, East Lansing, MI, 48823, USA

    Huanmin Zhang

Authors
  1. Zhihong Liao
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  2. Zhenkai Dai
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  3. Chenyu Cai
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  4. Xinheng Zhang
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  5. Aijun Li
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  6. Huanmin Zhang
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  7. Yiming Yan
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  8. Wencheng Lin
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  9. Yu Wu
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  10. Hongxin Li
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  11. Haiyun Li
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  12. Qingmei Xie
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Contributions

Zhihong Liao, Haiyun Li, Zhenkai Dai, and Qingmei Xie designed the study, analyzed the results, and revised the manuscript. Chengyu Cai, Xinheng Zhang, Aijun Li, and Huanmin Zhang performed the experiments and wrote the manuscript. Yiming Yan, Wencheng Lin, Yu Wu, and Hongxin Li analyzed the data and prepared figures.

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Correspondence to Qingmei Xie.

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Editor: Tetsuji Okamoto

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Liao, Z., Dai, Z., Cai, C. et al. Knockout of Atg5 inhibits proliferation and promotes apoptosis of DF-1 cells. In Vitro Cell.Dev.Biol.-Animal 55, 341–348 (2019). https://doi.org/10.1007/s11626-019-00342-7

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