Figure 3. Nerve growth factor promotes cell cycle progression via Akt and Erk activation in human corneal epithelial cells. A: Human corneal epithelial cells (HCECs) were incubated in keratinocyte serum-free medium (K-SFM) without growth factors for 24 h before treatment with nerve growth factor (NGF) at 25 ng/ml for 1 h. A total of 50 µg cell lysates and total RNA were analyzed for expression of the indicated genes by western blot analysis and reverse transcriptase–polymerase chain reaction (RT–PCR), respectively. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. B: HCECs were incubated in K-SFM without growth factors for 24 h and then pretreated with LY294002 or PD98059 at 10 µM for 1 h before treatment with NGF at 25 ng/ml for another hour. A total of 50 μg cell lysates were analyzed for expression of the indicated genes by immunoblotting analysis. GAPDH was used as a loading control. Experiments were performed in triplicate. C: The schematic representation depicts how the Akt and Erk pathways collaborate to control NGF induction of cyclin D expression.