Figure 5

Restoration of Nrf2 expression in transformed MSC induces an antioxidant response and impairs tumor growth. (A) Relative mRNA expression of Nrf2 and selected antioxidants measured by qRT-PCR in tMSC over-expressing either empty vector or Nrf2. These genes include NQO1, GSTM2, glutamate-cysteine ligase modifier and catalytic subunit (GCLM and GCLC respectively), CAT, GPX7, glutathione reductase (GSR) and glutathione synthetase (GSS). (B) Total cell extracts of tMSC expressing either empty vector or Nrf2 were analyzed for expression of Nrf2 and its downstream target NQO1 by immunoblotting. Polyclonal antibodies were used for Nrf2 and GAPDH served as a loading control. (C) Levels of intracellular reduced glutathione (GSH) presented as relative absorbance units per microgram of protein x10⁵. The result shows the average of 2 experiments performed with at least 4 replicates each. (D) Intracellular ROS levels of tMSC expressing either empty vector or Nrf2, measured by CM-H₂DCFDA staining. ROS amounts are presented as the geometrical mean fluorescence intensity (± SD) from at least three experiments. (E) Growth rate of tMSC expressing empty vector or Nrf2 at 5, 24 and 48 hours. The number of viable cells was expressed as relative absorbance units (R.A.U.). The average of three experiments with at least four replicate wells per cell line and normalized by the amount of protein is shown. (F) Soft agarose colonies of tMSC over-expressing empty vector or Nrf2 were photographed after 12 days in culture at x40 magnification (top) and counted in triplicate wells (bottom). (G) Comparison of animal survival between the Nrf2 over-expressing group and empty vector group containing six mice each (P = 0.001). P values are *P < 0.05 and **P < 0.005.