Forced Overexpression of Claudin-3 but not of Claudin-1 Increases the Malignant Potential of HT-29 Cells sellckchem We observed that EGF treatment did not cause statistically significant changes in claudin-1 expression but increased claudin-3 expression concomitantly with the malignant potential in HT-29 cells (Fig. 1B, ,3,3, ,44 and and5).5). Based on these findings, we further examined the effects of the forced claudin-1 and claudin-3 overexpression on cell migration, proliferation and colony formation. Interestingly, we observed that the overexpression of claudin-1 but not that of claudin-3 decreased cell migration (Fig. 7A and 7B). Conversely, cell proliferation was not altered by the overexpression of either claudin at the same time point (Fig. 7C).

Furthermore, we observed increases in the anchorage-dependent and anchorage-independent colonies formation after the overexpression of claudin-3 but not claudin-1 (Fig. 8 and and9,9, respectively). Because claudin-3 overexpression increased the colony formation in HT-29 cells, we assessed whether the forced overexpression of claudin-3 could increase cell proliferation at later time points (24, 48 and 72 h). We verified that claudin-3 overexpression increased cell proliferation at these times (Fig. S2), which could contribute to the increased malignant potential of HT-29 cells. These results indicate that the differential expression of claudins 1 and 3 plays a crucial role in the malignant phenotype of the HT-29 colorectal cancer cells. Figure 7 The effect of claudin-1 and claudin-3 overexpression on migration and proliferation.

Figure 8 The impact of claudin-1 and claudin-3 overexpression on anchorage-dependent colony formation. Figure 9 The impact of claudin-1 and claudin-3 overexpression on anchorage-independent colony formation. Claudin-3 Silencing Prevents the EGF-induced Malignant Potential of HT-29 Cells Because EGF treatment increased claudin-3 expression (Fig. 1B), and the overexpression of this claudin is related to the increased malignant potential of HT-29 Cilengitide cells (Figs. 8 and and9),9), we investigated whether the downregulation of claudin-3 could to prevent the EGF-induced effects in HT-29 cells using claudin-3 siRNA. Using immunoblot analysis, we confirmed a robust downregulation of claudin-3 24 h after transfection using both 25 and 45 nM of claudin-3 siRNA (Fig. 10A). Based on these results, we chose 25 nM of claudin-3 siRNA for subsequent functional analyses. Because claudin-3 overexpression-regulated colony formation is analyzed at late time points (more than 5 days), we evaluated whether the early downregulation of claudin-3 induced by siRNA (at 24 h) would be sufficient to prevent the events regulated by prolonged treatment with EGF.