Figure 4.

Effects of siRNA-based inhibition of claudin-1 expression in SW620 cells on proliferation, anchorage-independent growth, and invasion. (A) Results of soft agar assay. Experiments were performed as described in Methods. The number of soft agar colonies presented is the mean of colony counts from 3 different experiments. (B) A cell invasion assay was performed using 24-well transwells coated with collagen type I (100 μg/ml). After 72 hours of plating, cells from the top of the filter were removed, and the cells that invaded the coated membrane were fixed and counted. Data are presented as mean colony counts in ten ×20 microscopic fields from duplicate wells. *P < 0.05, as compared to control. (C) Gelatin zymography to determine the activities of MMP-2 and MMP-9 in SW620^control cells and in all 3 SW620^siRNA clones was performed as described in the Methods. (D) Cellular proliferation was measured in SW620^control and SW620^siRNA cells using MTT assay at 1, 3, 5, and 7 days after plating equal number of cells. (E) The anoikis assay was performed by plating the SW620^control and SW620^siRNA cells on polyHEMA-coated culture dishes for 72 hours as described in Methods. Values for control cells were considered 100%, and any differences are expressed relative to that value. Each bar represents the mean ± SD of 3 experiments. (F) The anoikis-induced apoptosis was quantitated using apoptosis-specific ELISA. Values for control cells were considered 100%, and any changes were compared with that value. Each bar represents the mean ± SD of 3 experiments.