Figure 6
Nrf2 sensitizes tMSC to apoptosis and diminishes the in vitro angiogenic response. (A) Apoptosis measured by flow cytometry after double staining with Annexin-V (FITC) and Propidium Iodide (PI). Left panel shows the percentage of cells in early apoptosis (positive for FITC) and late apoptosis (positive for FITC and PI) after 24 hours treatment with 5 μM camptothecin. Data show the mean ± S.D. from five independent experiments. Right panel shows the dot plots of a representative experiment. (B) Western-blot of nuclear and cytoplasmic extracts showing induction of cleaved PARP protein. Membrane was also probed with mouse monoclonal antibodies against Nrf2. Hsp90 and Lamin served as loading controls. (C) Caspase 3/7 activity of cells treated likewise. Results are expressed as relative absorbance units normalized by protein amount. Data show the mean ± S.D. from three independent experiments. (D) Western-blot shows destabilization of HIF-1α protein in Nrf2-over-expressing cells grown for 48 hours in hypoxia. Membranes were probed with mouse monoclonal antibodies for HIF-1α, Nrf2 and NQO1. HSP90 served as control. (E) qRT-PCR shows the VEGF-A fold induction in normoxic (21% O2) and 48 hours hypoxic (5% and 1% O2) cells. (F) Effect of hypoxic medium supernatant from tMSC over-expressing vector or Nrf2 in HUVEC viability. Supernatants from tMSC grown in hypoxia for 48 hours were mixed with endothelial cell medium at 50% ratio prior to incubation with HUVEC. A representation of three independent experiments shows the viable cells at 24, 48 and 72 hours as relative absorbance units. Western-blot shows the failure of Nrf2-expressing cells to accumulate HIF-1α protein in hypoxia (top-right). Increased NQO1 expression indicates activation of Nrf2 pathway. Equal loading of hypoxic tMSC medium supernatant in HUVEC is shown by coomassie blue staining (bottom-right). P values are *P < 0.05, **P < 0.005 and ***P < 0.0005.