G-CSF protected HRECs from oxidative stress-induced apoptosis through the PI3K-Akt pathway. (A) Left panel: The result of 24-hour exposure to 1mM H₂O₂; and the right panel, results of G-CSF addition. Note the decreased numbers of apoptotic cells (green squares) in the G-CSF-treated group. (B) Dose-dependent antiapoptotic effect of G-CSF. Cells were incubated with G-CSF (10, 100, or 1000 ng/mL) for 24 hours followed by exposure to 1mM H₂O₂ for another 24 hours. The maximum effect was observed with 100 ng/mL G-CSF (n = 6 at each point). *P < .05. (C) The rescue effect of 100 ng/mL G-CSF was attenuated by LY294002 (n = 6 at each point) but not by AG490. *P < .05. (D) Intracellular Jak2 (upper panel), stat3 (middle panel), and Akt (lower panel) phosphorylation profiles in G-CSF-activated HRECs. Lanes 1, 2, 9, and 10 are controls: lane 1, nonphosphorylated cell extracts; lane 2: phosphorylated cell extracts; lane 9: phosphorylated cell extracts; and lane 10: nonphosphorylated cell extracts. Lanes 3 to 8: HREC lysates, 20 μg per lane treated for 0 minutes (untreated, lanes 3 and 6), 10 minutes (lanes 4 and 7), and 30 minutes (lanes 5 and 8) with 100 ng/mL G-CSF. Only Akt was phosphorylated by G-CSF in HRECs, and the result was concordant with the results of the blocking experiments.