Notch1 inhibits cell invasion through CYFIP1.

<p>(a) SCC13 were starved for 2 days and then seeded in the matrigel coated chamber, where the coated membrane with 8 um pores separated the cells from the medium full in supplements. The cells were then cultured for 22 hours. After this timeframe the cells that migrated through the membrane were separated from the remaining cells and thus divided to invasive and non-invasive fraction of SCC13 cells. The cells were next stained for CYFIP1 and analyzed by FACS. The invasive fraction of SCC13 cells shows a lower percentage of CYFIP1 positive cells as compared to the non-invasive fraction (p < 0.001). (b) SCC13 cells were stably infected with a retroviral vector expressing a flag-tagged activated Notch1 protein fused to the human estrogen receptor (SCC13Nert), or empty vector control (SCC13Neo). The cells were subsequently treated with siRNA targeting CYFIP1 mRNA of unspecific control. The cells were next kept in starving conditions, seeded on the matrigel coated membrane with 8 um pores and treated with Tamoxifen. After 22h hours of incubation the invasive and non-invasive cells were counted and the invasion capacity was calculated as indicated in the manufacturer protocol. Section (b) represents the density of the invasive populations in the end of the experiment. (c) SCC13 with activated Notch1 and treated with control siRNA (Nert siCtrl) showed drastically decreased invasive capacity, which could be partially restored when the Notch1 activated SCC13 cells were treated with CYFIP1 specific siRNA (Nert siCYFIP1). (d) For control, the cells were parallel analyzed for CYFIP1 and p21 expression.