Alexa 488 and Alexa 594-conjugated secondary antibodies (Molecular Probes, Carlsbad, CA) were used. VX-770 Each specimen was visualized using a Zeiss LSM 510 META microscope (Carl Zeiss, Gottingen, Germany). siRNA-expressing constructs and the generation of stable cell lines The methods used were as previously reported [4]. We used a pSUPERgfp vector (OligoEngine, Seattle, WA) for expression of siRNA. The target sequences for the scrambled negative control and for BART were 5��-TTCTCCGAACGTGTCACGT-3�� and 5��-CATGGCAGCCTTCACCACA-3��, respectively. S2-013 and PANC-1 cells were transfected with either empty Neo-pSUPERgfp, a scrambled oligo-pSUPERgfp negative control, or a plasmid designed to express siRNA to BART, using FuGENE6 (Roche), according to the manufacturer’s instructions.

Cells were selected in medium containing 500 ��g/mL of geneticin to generate stable pSUPERgfp cell lines. Western blotting was performed to analyze the protein levels of the single clones. siRNA treatment RNAi targeting ANX7 and scrambled negative control siRNA oligonucleotides were purchased from Santa Cruz Biotechnology (29690 and 37007). For assays to examine the effect of siRNAs on ANX7 expression, S2-013 and PANC-1 cells that express ANX7 were plated in six-well plates. After 20 h, the cells were transfected with 80 pmols of siRNA in siRNA transfection reagent (Santa Cruz), following the manufacturer’s instructions. Transwell motility assay Cells (3.0��104) were plated in the upper chamber of BD BioCoat Control Culture Inserts (24-well plates, 8 ��m pore size; Becton Dickinson, San Jose, CA).

Serum-free culture medium was added to the upper chamber, and medium containing 5% FCS was added to the lower chamber. Cells were incubated on the membranes for 12 h, and motility was quantified. Matrigel invasion assay The two-chamber invasion assay was used to assess cell invasion (24-well plates, 8 ��m pore size, membrane coated with a layer of Matrigel extracellular matrix proteins; Becton Dickinson). Cells (4.0��104) were seeded in serum-free medium into the upper chamber and allowed to invade toward 5% FCS (the chemoattractant) in the lower chamber. After 20 h incubation, the number of invading cells at the bottom of the membrane was estimated under microscopic observation by counting three independent visual fields.

BART or ANX7 RNAi S2-013 cells were preincubated for 30 min in serum-free medium containing 250 nM Ro-32-0432, 10 mM safingol, 5.0 ��M rottlerin, or 5.0 ��M myristoylated PKC�� pseudosubstrate inhibitor. These preincubated cells were used Carfilzomib in the two-chamber invasion assay to assess cell invasion. Wound healing immunostaining assay A wound in the form of a cross was made through a confluent cell monolayer with a plastic pipette tip and cells were then allowed to polarize and migrate toward the wound. After 4 h, cells were immunostained with primary antibody and then incubated with fluorophore-conjugated secondary antibodies as described above.