Fig. 4. OA derivatives increase Nrf2 activity and its target gene expression. 24h after transfection with a reporter construct regulated by the antioxidant response element (ARE-luc), NIH3T3 cells were stimulated by PGJ2 (positive control), OA and its derivatives (A-Lac-O-Me, O-diol, Sc-O-ScIm). After next 24h luciferase was measured showing the increased activity of Nrf2 after stimulation by all compounds (A). Moreover, the effect of selected compound (A-Lac-O-Me) on Nrf2 protein level was checked using immunostaining analysis (B). 6h treatment with the OA derivatives led to the increased expression of HO-1 (gene regulated by Nrf2) as shown by real-time PCR (C). Nrf2 activation by triterpenoids might be cell-type dependent. After transfection of human endothelial cells (HMEC-1) and human keratinocytes (HaCaT) with ARE-luc plasmid and stimulation with three different concentrations of betulin, the increase in luciferase activity was observed only in HaCaT cells (D). (A, C, D) - mean of 3 independent experiments, *p<0.05, B - representative immunostaining for Nrf2 (green). DAPI staining was used to visualize nuclei (blue).