Fig. 2
PI3-K/Akt activity modulates cell migration. A, B EGFP- and EGFP-K-Ras(V12)-expressing PANC-1 cells were incubated until confluency and proliferation was inhibited by mitomycin C. A wound of 250–300 μm was scratched into the monolayer (time point t0) and cells were incubated in serum-free medium with 10 μM LY294002 [LY] or solvent [Control, Co.]. Cell migration was documented after 24 h and 48 h at three randomly chosen positions. Images in A show representative phase contrast pictures at t0 and t48. The Western blot on the right shows the detection of pAktSer473 in lysates 48 h after wounding. Detection of ERK2 served as loading control. B-D Quantification and statistics of independent wounding assays. The size of the wound was measured and calculated as percent wound closure at t48. The bar graph shows the mean ± SD, the dots represent individual data points (triplicate values (B + C) or 3–5 values (D), t-test (unpaired, two-tailed), *** p ≤ 0.001; **** p ≤ 0.0001). B Bar graph of the quantification of the wound closure of 3 independent experiments (triplicate values) as described above. C Bar graph of the quantification of 4–5 independent (triplicate values) in which cells were transfected with 66 nM siAkt targeting Akt1 and Akt2. Control cells [Co.] were treated with 66 nM scrambled siRNA (siControl). Wounding was started 48 h after transfection. Quantification was performed as described above. The Western blot on the right shows the detection of pAktSer473, total Akt, and ERK2 as loading control in lysates 48 h after wounding. D Bar graph presenting the quantification of wounding assays with EGFP- and EGFP-K-Ras(V12)-expressing Colo-699 cell clones performed in DMEM with 5% FCS for 48 h. Quantification was conducted as described above