Reactive oxygen species (ROS) are important regulators of cellular functions. In embryonic stem cells, ROS are suggested to influence differentiation status. Regulated ROS formation is catalyzed primarily by NADPH-dependent oxidases (NOXs). Apocynin and diphenyleneiodonium are frequently used inhibitors of NOXs; however, both exhibit uncharacterized effects not related to NOXs inhibition. Interestingly, in our model of mouse embryonic stem cells we demonstrate low expression of NOXs. Therefore we aimed to clarify potential side effects of these drugs. Both apocynin and diphenyleneiodonium impaired proliferation of cells. Surprisingly, we observed prooxidant activity of these drugs determined by hydroethidine. Further, we revealed that apocynin inhibits PI3K/Akt pathway with its downstream transcriptional factor Nanog. Opposite to this, apocynin augmented activity of canonical Wnt signaling. On the contrary, diphenyleneiodonium activated both PI3K/Akt and Erk signaling pathways without affecting Wnt. Our data indicates limits and possible unexpected interactions of NOXs inhibitors with intracellular signaling pathways.

Department of Histology and Embryology Faculty of Medicine Masaryk University Kamenice 753 5 62500 Brno Czech Republic

Institute of Biophysics Academy of Sciences of the Czech Republic Královopolská 2590 135 61200 Brno Czech Republic; International Clinical Research Center Center of Biomolecular and Cellular Engineering St Anne's University Hospital Pekařská 53 65691 Brno Czech Republic

Institute of Experimental Biology Faculty of Science Masaryk University Kotlářská 267 2 61137 Brno Czech Republic

Institute of Experimental Biology Faculty of Science Masaryk University Kotlářská 267 2 61137 Brno Czech Republic; Institute of Biophysics Academy of Sciences of the Czech Republic Královopolská 2590 135 61200 Brno Czech Republic; Department of Histology and Embryology Faculty of Medicine Masaryk University Kamenice 753 5 62500 Brno Czech Republic

Institute of Experimental Biology Faculty of Science Masaryk University Kotlářská 267 2 61137 Brno Czech Republic; International Clinical Research Center Center of Biomolecular and Cellular Engineering St Anne's University Hospital Pekařská 53 65691 Brno Czech Republic

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