Fig. 1. AU-565 cells migrate through an endothelial cell monolayer. (A) AU-565 cells were added to confluent BLMVEC monolayer in defined medium. Cells were washed and fixed to the coverslips 4 or 24 h later, stained with Texas-Red phalloidin to detect F-actin, and visualized by fluorescence microscopy. Images show distinct endothelial retraction (red arrowheads) and AU-565 cells (yellow arrows) in the process of transendothelial migration. (B) Representative ECIS plot showing resistance measurements of the BLMVEC monolayer before and after the addition of defined medium (dark green lines), additional BLMVEC (light green lines), AU-565 cells transfected with an empty pcDNA3.1 vector (light purple lines), or untreated AU-565 cells (dark purple lines). Measurements from both arrays are shown on the same plot. Thick lines represent measurements taken from Array A, and thin lines represent measurements taken from Array B. To account for variations in resistance measurements in different wells after medium swap, resistance is normalized by dividing resistance by the average monolayer resistance over approximately 30 min after monolayer recovery and before addition of cancer cells. ECIS measurements show a sharp drop in resistance following addition of untreated AU-565 cells to the BLMVEC monolayer, as do AU-565 cells transfected with the empty vector. Addition of more BLMVEC to the BLMVEC monolayer does not affect monolayer resistance. (C) Compilation of ECIS measurements at 5 h increments from multiple experiments shows that differences in monolayer resistance in wells containing AU-565 cells transfected with the empty vector (light purple squares) are statistically significant (p  0.05) 10 and 15 h following their addition to the monolayer, compared to untreated AU-565 cells (dark purple diamonds). Results are shown as fold decrease of resistance in sample wells compared to average resistance of the untreated BLMVEC monolayer. Error bars show standard error means. ^*p  0.05, ^**p  0.01, ^***p  0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2. RNA interference of FAK causes decreased transendothelial migration of AU-565 cells. (A) Representative ECIS plot showing resistance measurements of BLMVEC monolayer upon addition of defined medium (green lines), untreated AU-565 cells (dark purple lines) or AU-565 cells transiently transfected with Validated Silencer™ siRNA against FAK (red lines) or a negative control siRNA (light purple lines). AU-565 cells treated with siRNA show decreased transendothelial migration, whereas cells transfected with negative control siRNA behave similarly to untreated AU-565 cells. (B) Compilation of ECIS measurements at 5 h increments from separate experiments shows significant differences in monolayer resistance in wells containing untreated AU-565 cells (purple diamonds) versus wells containing siRNA-treated AU-565 cells (red squares). (C) Compilation of ECIS measurements at 5 h increments shows that differences in monolayer resistance in wells containing untreated AU-565 cells (purple diamonds) versus wells containing negative siRNA-treated AU-565 cells (light purple squares) are not significant. (D) Real-time RT-PCR results show approximately 50% knockdown of FAK mRNA in AU-565 cells that were transiently transfected with Validated Silencer™ siRNA for 72 h. FAK mRNA expression in AU-565 cells transfected with the negative control siRNA is unchanged compared to untreated AU-565 cells. ^*p  0.05, ^**p  0.01, ^***p  0.001. (E) Representative immunoblot of total FAK or GADPH levels in untreated cells (lane 1), cells transfected with a negative control siRNA (lane 2), and cells transfected with two different siRNA constructs (lanes 3 and 4). Histograms of the relative density of these bands are shown at bottom (n = 3; ^*p  0.01 relative to untreated cells). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3. FRNK inhibition of FAK causes decreased transendothelial migration of AU-565 cells. (A) Representative ECIS plot showing resistance measurements of BLMVEC monolayer upon addition of defined medium (green lines), untreated AU-565 cells (purple lines) or AU-565 cells expressing wildtype FRNK (red lines). ECIS resistance measurements of monolayer upon addition of AU-565 cells expressing FRNK show decreased transendothelial migration compared to untreated cells. (B) Compilation of ECIS measurements at 5 h increments from multiple experiments shows significant differences in monolayer resistance in wells containing AU-565 cells expressing FRNK. (C) Top panel: RT-PCR results showing expression of HA-tagged FRNK in stably transfected AU-565 cells. Bottom panel: FAK mRNA expression in both untreated and transfected cells is equivalent. ^*p  0.05, ^**p  0.01, ^***p  0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4. Overexpression of wildtype FAK does not increase AU-565 cell transendothelial migration. (A) Representative ECIS plot showing resistance measurements of BLMVEC monolayer upon addition of defined medium (green lines), untreated AU-565 cells (purple lines) or AU-565 cells over-expressing wildtype FAK (distinct clones designated wt FAK #7 and #8, shown in red and dark red lines, respectively). Measurements from both arrays are shown on the same plot. Thick lines represent measurements taken from Array A, and thin lines represent measurements taken from Array B. AU-565 cells over-expressing wildtype FAK show slightly decreased transendothelial migration within 15 h of addition to the BLMVEC monolayer. After 15 h, transendothelial migration of these cells compared to untreated AU-565 cells. (B) Compilation of ECIS measurements at 5 h increments from multiple experiments shows statistically significant differences in monolayer resistance in wells containing AU-565 cells over-expressing wildtype FAK (clone #7, red squares; clone #8, dark red triangles), versus wells containing untreated AU-565 cells (purple diamonds) from 5 to 20 h following addition of cancer cells to the monolayer. ^*p  0.05, ^**p  0.01 (C) RT-PCR results (top panel) show expression of myc-tagged wildtype FAK in stably transfected AU-565 cells. Untreated AU-565 cells (lane 1) and AU-565 cells transfected with the empty pcDNA3.1 vector (lane 2) do not express myc-tagged wildtype FAK, while cells transfected with wildtype FAK (clone #7, lane 3; clone #8, lane 4) do. Lower panel shows amplification of total FAK mRNA in each population. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)