Supplemental figures from Spi-B–Mediated Silencing of Claudin-2 Promotes Early Dissemination of Lung Cancer Cells from Primary Tumors

Figure S1, related to Figure 3. Effect of Spi-B on EMT. (A) Phase-contrast micrographs of HBECs expressing vector or Spi-B. Scale bars are 100μm; (B) western blot showed expression of Spi-B, E-cadherin, Vimentin and Actin; (C) Quantitative RT-PCR assay for Spi-B, Snail1, Snail2, Twist1 and ZEB1. Figure S2, related to Figure 3. (A) Phase-contrast micrographs of H526 cells expressing control or Spi-B shRNAs. Scale bars are 50μm; (B) BrdU in corporation assay of A549 expressing vector or Spi-B; (C) vector or Spi-B-expressing A549 cells were plated on fibronectin-coated plates. After 15 min, attached cells were counted. Scale bars represent 200 mm. Bar graph below shows number of adherent cells. Mean {plus minus} SD of three duplications is shown. Figure S3, related to Figure 5. Expression of claudin-2 in tumor-adjacent tissues. (A) IHC showed expression of claudin-2 in bronchial epithelial cells; (B) IHC showed expression of claudin-2 in alveolar epithelial cells (arrows). Scale bars are 50μm. Figure S4, related to Figure 6. Other Spi-B downregulated tight junction proteins, Claudin-14 and Crumbs3, are not able to rescue Spi-B induced cellular behavior. (A) A549 cells were transduced with Spi-B or Spi-B and Claudin-14 or Spi-B and Crumbs3 as indicated. Representative images of the clones in soft agar were shown in left panels. The bar chart shows the percentage of mass (blue), and loose colonies (red). Error bars indicate means {plus minus} SD for a representative experiment performed in triplicate. Scale bars represent 100 μm. (B) A549 cells were transduced with Spi-B or Spi-B and Claudin-14 or Spi-B and Crumbs3 as indicated. Phase contrast of acini were shown in left panel and quantified in right panel. The bar chart shows the percentage of mass (blue) and grape-like (red). Error bars indicate means {plus minus} SD for a representative experiment performed in triplicate. Scale bars represent 25 μm. (C) A549 cells were transduced with Spi-B or Spi-B and Claudin-14 or Spi-B and Crumbs3 as indicated and subjected to an invasion assay. Error bars indicate means{plus minus} SD for a representative experiment performed in triplicate. Scale bars represent 100 μm. Figure S5. (A) A549 cells were transduced with Spi-B or Spi-B and shRNA of MMP9 as indicated and subjected to an invasion assay. (B) A549 cells were transduced with Spi-B or Spi-B and MMP9 inhibitor (TIMP1) as indicated and subjected to an invasion assay. Error bars indicate means{plus minus} SD for a representative experiment performed in triplicate. Scale bars represent 100 μm.