H₂O₂ induced apoptosis and increased G-CSF and G-CSFR expression in HRECs. (A) Representative images of H₂O₂-induced apoptosis in HREC culture. Note that the number of apoptotic cells increased with time. h indicates hours after exposure to H₂O₂. (B) The number of apoptotic cells was measured by fluorescence-activated cell sorter using annexin V antibody. Increases in the number of annexin V-positive cells at 24 hours (green square) were observed. Note that numbers of both the early apoptotic cells (bottom right quadrant) and the late apoptotic or necrotic cells (top right quadrant) were increased by H₂O₂. (C) G-CSFR expression on HRECs was confirmed by Western blot analysis. (D) The effect of oxidative stress on G-CSFR or G-CSF mRNA expression in HRECs. Expression of G-CSF mRNA was significantly increased over time by oxidative stress (n = 4 at each point). **P < .01. Increased G-CSFR mRNA expression was observed at 6 hours after 1mM H₂O₂ exposure (n = 4 at each point), and the level was returned to normal at 24 hours. *P < .05.