Inhibition of mTORC2-AKT and ERK signaling pathway reduced T24 cells and E6 cells cell viability, migration and invasion in hydronephrotic urine treatment.

<p>To confirm the importance of mTORC2-AKT and ERK signaling pathway in T24 and E6 cells after the treatment of hydronephrotic urine, we used LY294002 (PI3K inhibitor) and PD184352 (ERK inhibitor) to analysis the cell function. (A) T24 cells and E6 cells were cultured in serum free medium and treated with LY294002 (20µM) and PD184352 (10µM) for 60 min and stimulated with 20µl/ml hydronephrotic urine from UUO 3 weeks for 30 min. The phosphorylation of mTOR-Ser2481, AKT-Ser473 and ERK were detected by western blotting (D: DMSO, LY: LY294002, PD: PD184352). (B, C) T24 cells and E6 cells were cultured in serum free medium and treated with LY294002 (20µM) and PD184352 (10µM) and stimulated with 20µl/ml hydronephrotic urine from UUO 3 weeks. The cells would be counted the cell number after treatment for 0, 24 and 48 hrs, respectively (Control: without hydronephrotic urine treatment, LY: LY294002, ERKi: PD184352). (D, E) T24 cells and E6 cells were cultured in serum free medium and treated with LY294002 (20µM) and PD184352 (10µM) and stimulated with 20µl/ml hydronephrotic urine from UUO 3 weeks for 24 hrs. The cells would be analyzed the migration capability by wound healing assay (Control: without hydronephrotic urine treatment, LY: LY294002, ERKi: PD184352). (F, G) T24 cells and E6 cells were cultured in serum free medium and treated with LY294002 (20µM) and PD184352 (10µM) and stimulated with 20µl/ml hydronephrotic urine from UUO 3 weeks for 12 hrs. The cells would be analyzed the invasion capability by transwell motility assay (Control: without hydronephrotic urine treatment, LY: LY294002, ERKi: PD184352).