International Journal of Molecular Sciences
The study was aimed at identifying endogenous proteins which assist or impede the permeabilized state in the cell membrane disrupted by nsEP (20 or 40 pulses, 300 ns width, 7 kV/cm). We employed a LentiArray CRISPR library to generate knockouts (KOs) of 316 genes encoding for membrane proteins in U937 human monocytes stably expressing Cas9 nuclease. The extent of membrane permeabilization by nsEP was measured by the uptake of Yo-Pro-1 (YP) dye and compared to sham-exposed KOs and control cells transduced with a non-targeting (scrambled) gRNA. Only two KOs, for SCNN1A and CLCA1 genes, showed a statistically significant reduction in YP uptake. The respective proteins could be part of electropermeabilization lesions or increase their lifespan. In contrast, as many as 39 genes were identified as likely hits for the increased YP uptake, meaning that the respective proteins contributed to membrane stability or repair after nsEP. The expression level of eight genes in different types of human cells showed strong correlation (R > 0.9, p < 0.02) with their LD50 for lethal nsEP treatments, and could potentially be used as a criterion for the selectivity and efficiency of hyperplasia ablations with nsEP.
© 2023 by the authors.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution 4.0 International (CC BY 4.0) license.
Article states: The data presented in this study are available on request from the corresponding author.
Original Publication Citation
Silkuniene, G., Mangalanathan, U. M., Rossi, A., Mollica, P. A., Pakhomov, A. G., & Pakhomova, O. (2023). Identification of proteins involved in cell membrane permeabilization by nanosecond electric pulses (nsEP). International Journal of Molecular Sciences, 24(11), Article 9191. https://doi.org/10.3390/ijms24119191
Silkuniene, Giedre; Mangalanathan, Uma; Rossi, Alessandra; Mollica, Peter A.; Pakhomov, Andrei G.; and Pakhomova, Olga N., "Identification of Proteins Involved in Cell Membrane Permeabilization by Nanosecond Electric Pulses (nsEP)" (2023). Bioelectrics Publications. 356.
0000-0001-8436-9718 (Silkuniene), 0000-0003-0126-5660 (Mangalanathan), 0000-0001-9042-3549 (Rossi), 0000-0002-0621-2819 (Mollica), 0000-0003-3816-3860 (Pakhomov), 0000-0003-4950-4130 (Pakhomova)