VEGFA Upregulates FLJ10540 Figure 6. FLJ10540 alone or increased FLJ10540, mediated by VEGF-A stimulation, not only modulates cell migration and invasion through the PI3K/AKT signaling pathway, but also reinforces PI3K complex formation. (A) Vehicle-CL1-0, CL1-0-FLJ10540, vehicle-RK3E, and RK3E-FLJ10540 infected cells were serum-starved for 24 hours and treated with or without the indicated inhibitors, including SB202190, PD98059, and LY294002 for 2 hours. The migration and invasion ratios of vehicle-CL1-0, CL1-0-HA-FLJ10540, vehicle-RK3E, and RK3E-Flag-FLJ10540 infected cells were determined as previously described. (B) CL1-0 and RK3E-expressing FLJ10540 cells were treated with or without LY294002 at the final concentration of 10 mM. The total cell lysates were subjected to immunoblot analysis for the unphosphorylated or phosphorylated form of AKT. b- actin was used as an internal loading control. (C) CL1-0 cells expressing HA-FLJ10540 were pre-treated with or without VEGF-A (20 ng/ml). After 10 min, LY294002 (10 mM) was added and cells were further incubated for 2 hours. The total cell lysates were subjected to immunoblot analysis for HA, FLJ10540, or the unphosphorylated or phosphorylated forms of AKT. (D) A negative control siRNA and two different FLJ10540 siRNAs (siFLJ10540- PLoS ONE | www.plosone.org 9 April 2009 | Volume 4 | Issue 4 | e5052