Figure 3. APIP affects both AKT and ERK1/2 pathways for cell proliferation.

A. and B. Knockdown or overexpression of APIP regulates AKT and ERK1/2 phosphorylation. Whole-cell extracts of SNU-16 control and APIP knockdown cells (A) or of SNU-620 control and APIP overexpression cells (B) were analyzed by western blotting. C. Reconstitution of APIP* rescues AKT and ERK1/2 phosphorylation in APIP knockdown SNU-16 cells. SNU-16 control and APIP knockdown cells were transfected with pcDNA, pAPIP or pAPIP* for 36 h. D. Effects of RAF1-MEK inhibition on cell proliferation in SNU-16 APIP knockdown cells. SNU-16 control and APIP knockdown cells were treated with 2 μM U0126 or 1 μM AZ628 for 4 days. E. Altered cell cycle distributions by APIP knockdown in asynchronous culture. SNU-16 control and APIP knockdown cells were stained with propidium iodide and analyzed by flow cytometry. Numbers indicates cell percentages at G₁ phase. F. APIP knockdown decreases the expression level of cyclin D1. Asynchronous SNU-16 control and APIP knockdown cells were analyzed by western blotting. G. Overexpressed APIP exhibits focus-forming activity through MEK1/2 and PI3K. NIH3T3 control and APIP-overexpressing cells were assayed for focus formation with 2 μM U0126, 10μM LY294002 or 5 ng/ml rapamycin for 14 days, as visualized by crystal violet. Representative plates (left) and quantification of foci (right) are shown. Scale bar, 1 cm. H. Enhanced phosphorylation of AKT and ERK1/2 and cell growth in the primary APIP transgenic MEFs. Primary MEFs from wild-type (#1) and APIP transgenic (#1) were analyzed by western blotting (left) or maintained for 4 days to measure cell growth rate (right). All data are represented as mean ± S.D. (n = 3). Statistical significance is indicated as follows: **, P < 0.01. ***, P < 0.005.