Characterization of AGR-2-silenced PC3 cells.

<p><b>A</b>. MTT cell proliferation assay showing similar rates of growth in PC3^control and PC3^AGR2sh cells. <b>B</b>. PC3 ^control cells (above) showing a fibroblast-like appearance with pseudopodia-like extension for cell-cell contacts. PC3^AGR2sh cells (below) showed a more epithelial cell-like, rounded phenotype. These cells were also loosely attached to the culture dishes as compared to control cells. The green fluorescence in the right panels shows both PC3^control and PC3^AGR2sh cells constitutively express GFP because of the presence of GFP expressing cassette in the vectors used to create the control and AGR-2-silenced PC3 cell lines.</p

A. Wound Closure Assay.

<p>High AGR-2 expressing PC3 ^control cells and low AGR-2 expressing PC3 ^AGR−2sh cells were grown in 6 well culture dish up to 90% confluency. They were serum starved for overnight and allowed to migrate following creation of wound using a 200 µl pipette tip and adding fresh complete medium. Photographs were taken at 0 hr and 22 hrs for determination of rate of wound closure between the two cell lines. <b>B</b>. <b>Boyden Chamber Assay</b>: PC3 ^control cells and PC3 ^AGR−2sh cells were plated on cell culture inserts in serum free medium and migration of cells was stimulated adding 10% FBS as chemoattractant in the bottom chamber. After 6 hrs of incubation, cells were removed from the top of the insert using a cotton swab and cells on the underside of the insert were photographed and counted. The effect of AGR-2 silencing on PC3 cell migration is presented here as relative values compared to control (100%).</p

Cell adhesion properties and integrin expression of AGR-2-silenced PC3 cells.

<p><b>A.</b> Adhesion properties of PC3^control and PC3^AGR2sh cells were evaluated following growth in culture dishes coated with various ECM proteins. First, cells were seeded in duplicate onto the coated substrates and allowed to adhere. Next, unbound cells were washed away, and the adherent cells were fixed and stained. Finally, the stain was extracted and quantified colorimetrically. Significant reduction in cellular adhesion to fibronectin (***p<0.001), collagen I (**p<0.01), collagen IV (***p<0.001), laminin (*p<0.05) and fibrinogen (*p<0.05) were observed in case of PC3^AGR2sh cells when compared to PC3^control cells. BSA coated wells were used as the reagent control (p>0.1). Data presented here are mean ± SEM. <b>B</b>. Significant down-regulation of α4, α5, αV, β3 and β4 integrins was observed in PC3^AGR2sh cells as compared to PC3^control cells. No difference in β1 integrin levels was observed between the two cell types. Beta-actin was used as the loading control. Multiple protein bands present in the blots include integrin precursor and mature proteins as well as cleaved products is expected molecular mass according to manufacturer’s information (Cell Signaling Technology, Cat# 4749S).</p