Figure 2

Invasion assay: 1 × 10⁵ cells were added to the upper compartment of the chamber. Growth factors added in the medium (complete medium) were used as chemoattractant and placed in the lower chamber. After incubation for 23 h at 37°C in a 5% CO₂ incubator, the cells on the upper surface of the filter were completely removed by wiping with a cotton swab, and the cells that had crossed the matrigel and attached to the lower surface of the filter were counted. The filters were fixed, stained with Diff Quick (Sigma), cut out, and mounted on glass slides. The three fields of cell number that crossed the membrane were counted under a light microscope and the average value was calculated. Experiments were performed three times with four chambers/cell line. Bars represent ± SD, P < 0.05 (A). In vitro wound-healing assay: the wound was created by scraping with loading tips. Images were captured 18 h after adding the growth factors in both SAEC and SAEC-A0.5 cells. The wound-closing trend was compared between the two types of cells upon addition of the growth factors (GF) (B). Surface expression of integrin receptors on arsenic-treated SAEC cells and controls. Fluorescence flow-cytometry analysis of surface expression of two endogenous integrins, the laminin receptor anti-α2 and the collagen receptor anti-β4, in arsenic-treated and untreated SAEC cells determined by staining with the correspondent antibodies, respectively (C). Rate of lamellipodia formation was compared between SAEC and SAEC-A0.5 cells by scoring the number of cells that actively form lamellipodia (D). Five hundred cells in three different coverslips were scored and recorded under a fluorescence microscope; percentage of cells with lamellipodia was calculated. Bars represent ± SD, P < 0.01 (D). Fluorescence microscopy of phalloidin stained SAEC and SAEC-A0.5 cells (E). Cells were grown on glass coverslips for 24 h, fixed, and labeled with Rhodamine-phalloidin to visualize actin filaments. Arrows point to lamellipodia areas. Magnification 600×.