FIG 9.

Notch expression in PAE cells protects from apoptosis. (A) FACS analysis by annexin V-APC and propidium iodide (PI) staining at 16 h after adding 200 μM hydrogen peroxide (H₂O₂) to CTL and NICD2 cells. PI⁻/annexin V⁺ cells (Q4) were considered early apoptotic. Representative results from one of the three experiments (which all gave similar results) are shown. (B and C) Total protein extracts of indicated cells, treated where indicated with H₂O₂ for 24 h, were analyzed by Western blotting. A representative experiment is shown (C), as well as the quantification of cleaved caspase-3 (B), using the averages ± the SD (n = 4). (D) Active levels of Akt1 and Akt2 were analyzed as described for Fig. 3D. (E and F) Changes in FoxO1 were determined in control (Ctl) and NICD2-expressing cells by RT-qPCR (E) or by Western blotting (F). Phosphorylated FoxO1 (pS256-FoxO1) was also determined (F). (G and H) The quantification of three different Western blot analyses. *, P < 0.05 compared to CTL.