EGF neither induces E2F4 phosphorylation and nuclear translocation nor G1/S phase transition in HIEC. A. Subconfluent HIEC were serum-deprived during 36 h, stimulated with 5% FBS or 100 ng/ml EGF or 10 μM LPA for 30 min, 4 h or 24 h. Equal amounts of whole cell lysates were separated by SDS-PAGE, and proteins were analyzed by Western blotting with specific antibodies against phosphorylated ERK1/2, pRb, cyclin D1, cyclin A, p27 and β-actin. B. Cells were also fixed after 24 h stimulation with 3% paraformaldehyde in PBS and permeabilized with 0.1% Triton X-100 for subsequent immunofluorescence staining of E2F4 and Ki67. Cells with nuclear E2F4 and Ki67 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, EGF or LPA stimulation are shown. Of note, each cell exhibiting nuclear E2F4 was positive for Ki67 staining. Results are the mean ± SEM of an experiment representative of 3. * Significant at p < 0.0001 compared to control cells (no serum) (Student’s t test). C and D. HIEC were serum-deprived during 36 h, stimulated with 5% FBS or 100 ng/ml EGF or 10 μM LPA for 30 min. Cell lysates were separated by 7.5% SDS-PAGE and proteins were analyzed by Western blotting with specific antibodies against E2F4 and β-actin.
Paquin et al. BMC Cell Biology 2013 14:33 doi:10.1186/1471-2121-14-33