Abstract
Ectopic gene expression is an indispensable tool in biology and medicine. However, it is often limited by the low efficiency of DNA transfection. It is known that depletion of p62/SQSTM1 enhances DNA transfection efficiency by preventing the degradation of transfected DNA. Therefore, p62 is a potential target of drugs to increase transfection efficiency. To identify drugs that enhance transfection efficiency, a non-biased high-throughput screening was applied to over 4,000 compounds from the Osaka University compound library, and their p62-dependency was evaluated. The top-scoring drugs were mostly microtubule inhibitors, such as colchicine and vinblastine, and all of them showed positive effects only in the presence of p62. To understand the mechanisms, the time of p62-dependent ubiquitination was examined using polystyrene beads that were introduced into cells as materials that mimicked transfected DNA. The microtubule inhibitors caused a delay in the ubiquitination. Furthermore, the level of phosphorylated p62 at S405, which is required for ubiquitination during autophagosome formation, markedly decreased in the drug-treated cells. These results suggest that microtubule inhibitors inhibit p62-dependent autophagosome formation. Our findings provide new insights into the mechanisms of DNA transfection and also provide a solution to increase DNA transfection efficiency.
Competing Interest Statement
The authors have declared no competing interest.
Abbreviations
- ATG
- autophagy-related gene
- CK2
- Casein kinase 2
- GAPDH
- glyceraldehyde 3-phosphate dehydrogenase
- GFP
- green fluorescent protein
- HDAC6
- histone deacetylase 6
- KO
- knockout
- LC3
- microtubule-associated protein light chain 3
- MEF
- murine embryonic fibroblast
- NF-κB
- nuclear factor-kappa B
- TBK1
- TANK-binding kinase 1
- ULK1
- Unc-51 like autophagy activating kinase1
- WIPI1
- WD repeat domain phosphoinositide-interacting protein 1
