β catenin

siRNA targeting human Caveolin 1 (39) β-catenin (35) were purchased from Dharmacon as a SMART pool mix of four sequences and PTEN (38) from Santa Cruz Biotechnologies as a mix of three sequences. Caveolin-1: 5′-GCA AAU ACG UAG ACU CGG A-3′, 5′-AUU AAG AGC UUC CUG AUU G-3′, 5′-GCA GUU GUA CCA UGC AUU A-3′ and 5′-CUA AAC ACC UCA ACG AUG A-3′. β-catenin: 5′-GAA CGC AGC AGC AGU UUG U-3′, 5′-CAG CUG GCC UGG UUU GAU A-3′, 5′-GCA AGU AGC UGA UAU UGA C-3′ and 5′-GAU CUU AGC UUA UGG CAA U-3′. si Scrambled, with no known human targets, was purchased from Invitrogen as a mix. Briefly, cells were transfected with 50 pmol of siPTEN, siCAV1, siβ-catenin or siScrambled (siScr) and were assayed for Luciferase activity or protein content 48 h post transfection.

Specific siRNA oligonucleotides against p70^S6K (5′-GACAAAAUCCUCAAAUGUA-3′), P-cadherin (5′-GAGGGUGUCUUCGCUGUAG-3′), β1 integrin (5′-CCACAGACA UUUACAUUAA-3′), ST6Gal-I (5′-ACUCAGAUAUCCCAAAGUG-3′), p120-catenin (5′-UAGCUGACCUCCUGACUAA-3′), β-catenin (5′-AAGUCCUGUAUGAGUGGG AAC-3′), and nonspecific duplex oligo (5′-GGCTACGTCCAGGAGCGCA-3′) were obtained from Dharmacon (Lafayette, CO, USA). Transfection was performed using siLentFect (Bio-Rad, Hercules, CA, USA) according to the manufacturer's procedures. To stably attenuate expression of p70^S6K, β1 integrin, and P-cadherin, we infected cells with lentivirus containing p70^S6K shRNA, β1 integrin shRNA or P-cadherin shRNA, or nonspecific shRNA as a control. Lentivrus-infected cells were selected for 72 hours in medium containing 1 μg/ml puromycin (Invitrogen, San Diego, CA, USA). To ectopically express active p70^S6K (D₃E-E₃₈₉) in CaOV-3 cells, we used a myc-tagged cDNA encoding the constitutively active p70^S6K (generously given by Dr. George Thomas, Genome Research Institute, University of Cincinnati, OH, USA).