Reduced expression of CD44 and CD155 following siRNA-KD in SNB-19 cells.

<p><b>a</b>, siRNA-KD of CD44 and CD155 was confirmed by Western blotting using both wild type (<i>wt</i>) and non-targeting siRNA treated cells as controls. GAPD-siRNA was used as a positive control to validate the knockdown effect. <b>b</b>, Expression of CD44 (<b>top panels</b>) and CD155 (<b>bottom panels</b>) in non-targeting siRNA treated cells (<b>control/left panels/L</b>) and relevant siRNA-KD (<b>right panels/R</b>) cells; scale bars: 25 µm. In control cells (<b>L</b>), CD44 is uniformly distributed with intense staining at the edges of the cells (arrows) whereas CD155 is well distributed with dense staining zones at the leading edges of the cells (arrows). CD44/CD155 staining was reduced in the siRNA-KD cells with clearly altered morphology (<b>R</b>). <b>c</b>, CD44/CD155-KD was confirmed by flow cytometry. Expression levels of CD44 and CD155 were significantly reduced in siRNA-KD SNB-19 cells as indicated by percentage of positive cells and fluorescence fold. * indicates statistical significance compared to non-targeting siRNA treated cells (control).</p

Expression and close proximity of CD44 and CD155 on GBM cells.

<p><b>a–c</b>, Co-staining of CD44 (red) and CD155 (green) on SNB-19 and UPAB cells; scale bars: 25 µm (<b>a</b>) and 10 µm (<b>b</b>–<b>c</b>). TIRF microscopy (<b>c</b>) clearly demonstrated the juxtaposition of CD44 and CD155. CD155 is mainly expressed on the processes and invadapodia (arrows). <b>d</b>, Significantly increased expression of CD44 (<b>top panel</b>) and CD155 (<b>bottom panel</b>) in GBM cells confirmed by flow cytometry, as assessed by the percentage of antigen-expressing cells and increase in fluorescence fold. * indicates statistical significance compared to CC-2565, a normal human astrocyte cell line.</p

CD44/CD155-depletion inhibited invasion but enhanced proliferation of SNB-19 cells.

<p>Wild type (untreated) SNB-19 cells were used as a control in the mAb-blocking experiments; non-targeting siRNA treated SNB-19 cells were used as a control in the siRNA-KD experiments. <b>a</b>, Significantly reduced invasion following mAb-blocking (<b>left panel</b>) and siRNA-KD (<b>right panel</b>) was shown compared to controls. All images have scale bars of 50 µm. <b>b</b>, Proliferation was markedly increased in mAb-blocking (<b>left panel</b>) and siRNA-KD (<b>right panel</b>) cells compared to controls. * indicates statistical significance.</p

Western blotting of Rho GTPases in CD44/CD155-KD SNB-19 cells.

<p>High levels of Cdc42, Rac 1/2/3, RhoA, RhoB and RhoC were detected in non-targeting siRNA treated SNB-19 cells (control) whereas those proteins of interest were downregulated in CD44/CD155-KD cells.</p

Decreased adhesive potential of SNB-19 cells following mAb-blocking (a) and siRNA-KD (b) of CD44/CD155.

<p>Five different human ECMs including collagen I, fibronectin, laminin, tenascin C and vitronectin were employed and BSA was used as the control substrate. For each ECM substrate, the adhesive potential of manipulated SNB-19 cells (<i>via</i> either mAb-blocking or siRNA-KD) was compared with the relevant control cells. Wild type SNB-19 cells served as a control in the mAb-blocking experiments whereas non-targeting siRNA treated SNB-19 cells were used as a control in the siRNA-KD experiments. * indicates statistical significance compared to control.</p

Expression of F-actin and integrins and their co-localisation with CD44/CD155 on GBM cells.

<p><b>a</b>, Co-staining of F-actin (green/<b>left panels/L</b>) or β₁-integrin (green/<b>right panels/R</b>) with CD44 (red/<b>top panels</b>) and CD155 (red/<b>bottom panels</b>) on wild type UPAB cells. <b>b</b>, Co-staining of α_vβ₁-integrin (green/<b>L</b>) or α_vβ₃-integrin (green/<b>R</b>) with CD44 (red/<b>top panels</b>) and CD155 (red/<b>bottom panels</b>) on wild type UPMC (CD44 staining) and non-targeting siRNA treated SNB-19 cells (CD155 staining). All images in <b>a</b> and <b>b</b> have scale bars of 25 µm. <b>c</b>, Western blotting showed reduced expression of F-actin and integrins (α_v, β1 and β₃) in CD44/CD155-KD SNB-19 cells when compared to non-targeting siRNA treated cells (control).</p

Reduced motility rate in CD44/CD155-KD SNB-19 cells.

<p>Non-targeting siRNA treated SNB-19 cells were used as a control. <b>a</b>, Velocity of cell movement (<b>right panel</b>) with cell tracking (<b>left panel</b>) analysed over a period of 72 h. The speed by which CD44/CD155-KD cells moved was significantly reduced. <b>b</b>, Total distance moved by cells (<b>right panel</b>) with cell tracking (<b>left panel</b>) over a period of 72 h. The distance travelled by CD44/CD155-KD cells was markedly decreased. Start position: white arrow; end position: black arrow. * indicates statistical significance compared to control.</p