antioxidants. Some studies have suggested an association between cell sensitivity to oxidative stress and stefin B, an endogenous inhibitor of cysteine cathepsins. In this research, we wanted to confirm the effect of stefin B on oxidative stress induced by H2O2 in a model of wild-type primary tumour cells MMTV-PyMT and in cells with the knocked-out gene for stefin B. Cells with the knocked-out gene for stefin B had a significantly higher proportion of dead cells after treatment with H2O2 as wild-type cells, however, we did not measure caspase activity. We have shown that treatment with H2O2 in cells with the knocked-out gene for stefin B rapidly raises reactive oxygen species and causes cardiolipin oxidation. The treatment also affected raising the pH in lysosomes, but inhibiting cathepsin with a cysteine protease inhibitor showed no effect; therefore, cathepsins most likely do not play an essential role in H2O2-induced cell death. The addition of the antioxidant N-acetyl-cysteine improved cell viability and limited the formation of ROS. The addition of the antioxidant vitamin C did not affect ROS formation and has even lowered cell viability. Based on these results, we concluded that not all antioxidants are equally effective and that their effectiveness depends on many factors (e.g., antioxidant concentrations used and ROS type). We can conclude that we did not trigger apoptosis in our cell model by H2O2 treatment but some other form of cell death. Activation of NLRP3 inflammasome and ROS-dependent cell death (e.g., ferroptosis) are most likely to occur in cells with absent stefin B, which could be confirmed in continuation the research task.
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