Figure 6
Inhibition of the p38 and ERK MAPK pathways alleviates the stimulatory effect of Rac1b knockdown on TGF-β1-induced migration. (A) Panc1 cells were transfected with siRNA specific for Rac1b (siRac1b) or an appropriate control siRNA (siCo). Transfected cells were serum-starved overnight and subjected to xCELLigence cell migration assay, in the presence of TGF-β1 (T, 5 ng/ml) and either SB203580 (10 µM) or vehicle (DMSO, 0.1%). Cell migration was measured every 15 min for 24 h. Letters to the right of each graph allow for a colour-independent identification of the various curves. Differences between siRac1b-transfected vehicle (green curve/tracing B) and SB203580 (magenta curve/tracing D) treated cells become significant as early as 2 h and remain so at all later time points. (B) Individual clones of Panc1 cells expressing MKK6KA or empty-vector control cells were transiently transfected with siRac1b or siCo. Transfected cells were serum-starved and subjected to xCELLigence cell migration assay in presence of 5 ng/ml TGF-β1. Cell migration was recorded every 15 min for 24 h and the results for two individual clones plotted against the control cells are shown. Differences between siRac1b-transfected vector cells (green curves/tracings B) and MKK6KA expressing cells (magenta curves/tracings D) from both clones become significant at the 22 h time point. (C) Panc1 cells transiently transfected with siRac1b or siCo and subsequently treated with TGF-β1 were analysed by real-time cell migration assay in the absence or presence of U0126 (10 µM). The difference between siRac1b-transfected vehicle-treated cells (green curve/tracing B) and U0126-treated cells (magenta curve/tracing D) is significant at the 10 h and all later time points. (D) As in (C), except that IMIM-PC1 cells were used. The difference between siRac1b-transfected cells treated with vehicle (green curve/tracing B) and those treated with U0126 (magenta curve/tracing D) is significant at the 8 h and all later time points. In each panel, a representative experiment is shown from three experiments performed in total with similar results. Note that for the sake of clarity, only the data from TGF-β1-treated cells are shown.
