Figure 4

puPA inhibits NICD and HES1-induced AKT, NF-κB and ERK phosphorylation in glioblastoma cells. Down regulation of uPA/uPAR inhibits AKT, NF-κB, ERK and mTOR pathway in (A) U251 MG (B) 5310 and (C) 4910 cells. pSV: scrambled vector; puPA: shRNA against uPA; puPAR: shRNA against uPAR and pU2: shRNA against both uPA and uPAR, DAPT-GSI (Gamma secretase inhibitor). puPA, puPAR and pU2 inhibits nuclear activation of p52, p65 and p50 in (D) U251 MG (E) 5310 and (F) 4910 cells. Nuclear extracts prepared from U251 MG, 5310 and 4910 cells which were left untransfected, transfected with pSV, puPA, puPAR, and pU2 and treated with DAPT and analyzed for nuclear activation of p52, p50 and p65 subunits of NF-κB using the Active Motif Transcription factor analysis kit following the manufacturer's instructions. puPA inhibits NICD and HES- induced AKT and ERK phosphorylation and NF- p65 in U251 MG and 4910 cells. Whole cell lysates from (G) U251 MG or from (H) 4910 xenograft cells over expressing NICD (Notch intracellular domain) or HES were transfected with pSV, transfected with puPA and were immunoblotted for pAKT, NF-κB p65 and pERK. Blots were reprobed with GAPDH to analyze for equal loading of proteins. Data represents average of triplicates normalized to GAPDH (**p < 0.01). (I & J) siRNA against Notch 1 inhibits phosphorylated forms of mTOR/ERK/AKT and NF- κB p65 in (I) U251MG and (J) 4910 cells. Whole cell lysates from U251MG and 4910 cells were left untreated, transfected with pSV and siNotch for 48 hrs and were immunoblotted for pmTOR, pERK, pAKT and NF- κB p65. A representative blot of three independent experiments is shown. Blots were reprobed with e GAPDH to analyze for equal loading of proteins. (K&L) shRNA against uPA, uPAR and U2 inhibited NICD/HES1 induced Notch 1 expression in U251MG and 4910 cells. (K) Whole cell lysates from (G) U251 MG or from (H) 4910 xenograft cells over expressing NICD (Notch intracellular domain) or HES were transfected with pSV, puPA, puPAR and pU2 and were immunoblotted for Notch 1 expression. A representative blot of three independent experiments is shown. Blots were reprobed with GAPDH to analyze for equal loading of proteins.