Stable knockdown of HIF-1α in SW480 cells results in decreased β-catenin expression levels and co-localization of β-catenin with E-cadherin at the cell membrane.

<p><b>A)</b> Upper panel: Decreased β-catenin expression in HIF-1α-knockdown SW480 cells. The cells were incubated in the presence of EDTA, washed, and incubated with mouse anti-β-catenin antibody. The cells were washed and stained with goat Alexa647-conjugated anti-mouse secondary antibody and examined by flow cytometry. Controls stained with only the secondary antibody were prepared and were used as controls (not shown). The figure shows the overlapping histograms of the labeled SW480 control cells (blue line and Ctr in the bar graph), HIF-1α-silenced cells (green line), and HIF-2α-knockdown cells (orange line). The bar graph on the right shows the means of the median fluorescence intensity ± SEM from at least four independent experiments. *: p<0.05. <b>Middle panel:</b> Quantification of β-catenin in nuclei isolated from SW480 control (scrambled shRNA) or HIF-1α-depleted cells. <b>Lower panel:</b> Quantification of β-catenin in nuclei isolated from SW480 control (scrambled shRNA) or HIF-2α-depleted cells. The nuclei were purified and labeled with anti-β-catenin and with Alexa 647-conjugated goat anti-mouse antibody in staining buffer as described in the “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112580#s2" target="_blank">Materials and Methods</a>”. After the final wash, the nuclei were fixed and analyzed by flow cytometry. Independent gates were generated for intact cells and for isolated nuclei stained with propidium iodide prior to the flow cytometry analysis. The figure shows the overlapping histograms of the labeled SW480 control cells (blue line and Ctr in the bar graph), HIF-1α-knockdown cells (green line), and HIF-2α-knockdown cells (orange line). A representative histogram from at least three independent experiments is shown. *: p<0.05; ***: p<0.001. <b>B)</b> and <b>C)</b> Stable control (scrambled shRNA) or HIF-1α- or HIF-2α- silenced SW480 cells were fixed, permeabilized, and co-immunostained with antibodies against β-catenin and E-cadherin (panel B) or against Snail 1 and vimentin (panel C). Nuclear staining was obtained by incubating the coverslips with 4′6-diamidino-2-phenylindol (DAPI). The fluorescence was analyzed by laser confocal microscopy as described in the “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112580#s2" target="_blank">Materials and Methods</a>”. E-cadherin and vimentin were visualized with Cy3-conjugated goat anti-rabbit antibody, and β-catenin and Snail 1 were visualized with Alexa 647-conjugated goat anti-mouse antibody. Controls were stained only with the secondary antibody, and no fluorescence signals were obtained from these controls (not shown). The data are representative of three independent experiments. Scale bar: 10 µm. D) Expression analysis of the EMT-related markers by western blot. Cytoplasmic and nuclear fractions were obtained from the cell extracts of control (Ctr in the figure) or HIF-1α- or HIF-2α- silenced SW480 cells using a nuclear isolation kit (Sigma), and the presence of the proteins indicated in the figure was analyzed in each fraction and in cell extracts by western blot. β-tubulin and lamin A/C were employed as a cytoplasmic marker and a nuclear marker, respectively, to confirm a lack of contamination and thus successful fractionation. The data are representative of three independent experiments.