Increased expression of NOX4 protein is associated with cancer progression and metastasis but the role of NOX4 in cell proliferation and invasion is not fully understood. We generated NOX4 knockout HeLa cell lines using the CRISPR-Cas9 gene editing system to explore the cellular functions of NOX4. After transfection of CRISPR-Cas9 construct, we performed T7 endonuclease 1 assays and DNA sequencing to generate and identify insertion and deletion of the NOX4 locus. We confirmed the knockout of NOX4 by Western blotting. NOX4 knockout cell lines showed reduced cell proliferation with an increase of sub-G1 cell population and the decrease of S/G2/M population. Moreover, NOX4 deficiency resulted in a dramatic decrease in invadopodium formation and the invasive activity. In addition, NOX4 deficiency also caused a decrease in focal adhesions and cell migration in HeLa cells. These results suggest that NOX4 is required for both efficient proliferation and invasion of HeLa cells.

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Published in PLOS ONE, v. 12, 1, e0170327, p. 1-13.

© 2017 Jafari et al.

This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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This study was supported by America Cancer Society Research Scholar Grant (RSG-13-184-01-CSM) and by the National Research Foundation of Korea (NRF) grant funded by the Korea government (NRF-2016R1A2B4009665).

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S1 Fig. Relative expression levels of NOX4 and p22phox.

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S2 Fig. NOX4 knockout did not affect the expression of cyclin D1 or caspase 3 cleavage.

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S3 Fig. Expression of NOX4 and p22phox are elevated in cervical cancers.