Figure 4. Distinct intracellular signaling pathways mediate apelin-induced proliferation, migration, and collagen I production. A: RPE cells were treated with various kinase inhibitors in the presence of 10⁻⁷ M apelin. Blocking apelin-induced RPE cell proliferation by pretreatment with 10 μM LY294002 and 20 μM PD98059 for 30 min, as measured with MTT assay after 48 h incubation. *p<0.05 versus untreated control. #p<0.05 versus apelin-treated alone. B, C: Inhibition of apelin-induced migration by pretreatment with 10 μM LY294002 and 20 μM PD98059 for 30 min, as measured with a transwell assay after 6 h incubation (a: control; b: 10⁻⁶ M apelin; c: LY294002+10⁻⁶ M apelin; d: PD98059+10⁻⁶ M apelin. 200× magnification). The number of migrated cells per HPF is shown. *p<0.001 versus untreated control. #p<0.01 versus apelin-treated alone. D: Complete inhibition of apelin-induced collagen I expression by 10 μM LY294002 and 20 μM PD98059. RPE cells were stimulated with 10⁻⁷ M apelin with or without pretreatment with kinase inhibitors for 30 min. After 3 h incubation, RNA was extracted, and collagen I expression was measured with real-time PCR. *p<0.05 versus untreated control. #p<0.05 versus apelin-treated alone. The data shown represent the mean±standard deviation (SD) of three independent experiments.