Figure 4
Chemical stabilization of Nrf2 induces antioxidant enzymes, reduces ROS, and impairs MSC transformation. (A) Nuclear and total cellular extracts of MSC3 and tMSC treated with 10 μM TBHQ or vehicle for 48 hours were analyzed for the expression of Nrf2, NQO1 or G6PD by immunoblotting. Rabbit monoclonal antibodies were used for Nrf2, and lamin and actin were used as loading controls. (B) qRT-PCR of Nrf2 and Nrf2-dowstream genes such as HO-1 and NQO1 was performed in tMSC treated for 48 hours with 10 μM TBHQ or vehicle control. Non-transformed MSC3 were used as a control. (C) Intracellular ROS concentration measured by CM-H2DCFDA staining in tMSC treated with or without 10 μM TBHQ for 48 hours. MSC3 ROS levels are also shown. ROS amounts are presented as the geometrical mean fluorescence intensity (± SD) from at least three experiments. (D) Cell viability after treatment with 10 μM TBHQ or vehicle control. A representation of three independent experiments is shown and data were calculated from the average of four replicate wells. Results are shown as percentage of viable cells referred to control cells (MSC3) treated with vehicle control. (E) Soft agarose colonies of tMSC treated with vehicle only or with 10 μM TBHQ were photographed at x40 magnification after 12 days in culture (top) and counted in triplicate wells (bottom). P values are *P < 0.05 and **P < 0.005.