Figure 5

GSK3 inhibition is required for phosphorylation and nuclear translocation of E2F4 as well as G1/S phase entry of HIEC. A. Subconfluent HIEC were serum-deprived during 36 h and then stimulated with 5% FBS or 100 ng/ml EGF or 10 μM LPA for 30 min and 24 h. Equal amounts of whole cell lysates were analyzed by Western blotting with specific antibodies against phosphorylated Akt, phosphorylated GSK3β and β-actin. B. Subconfluent HIEC were serum-deprived during for 36 h, stimulated with 5% FBS or 100 ng/ml EGF in presence or absence of 20 μM SB216763 (or DMSO) for 24 h. Equal amounts of whole cell lysates were separated by SDS-PAGE, and proteins were analyzed by Western blotting with specific antibodies against pRb, cyclin D1, p27 and β-actin. C. HIEC were serum-deprived during 36 h and then stimulated with 5% FBS or 100 ng/ml EGF for 30 min with or without prior 10-min 20 μM SB216763 (or DMSO) treatment. Cell lysates were analyzed by Western blotting with specific antibodies against E2F4, ERK2, phosphorylated ERK1/2, phosphorylated glycogen synthase (GS) and β-actin. D. Cells were also fixed after 24 h stimulation and permeabilized with 0.1% Triton X-100 for subsequent immunofluorescence staining of E2F4 and Ki67. Cells with nuclear E2F4 and positive for Ki67 staining were counted in 10 fields. Total cell number was determined using DAPI staining. Ratio of nuclear E2F4 expressing cells and Ki67 positive cells before/after serum or EGF +/− SB216763 (SB) stimulations are shown. Of note, each cell exhibiting nuclear E2F4 was positive for Ki67 staining. Results are the mean ± SEM of 2 separate experiments. * Significant at p < 0.05. ** Significant at p < 0.01. *** Significant at p < 0.005 compared to control cells (no serum) (Student’s t test).