Dfc500

RKO and HCT116 infected cells were spread at the bottom of 96-well plates. A linear wound was created by scraping with a 100-µl pipette tip across the confluent cell layer. After washing the cells three times to remove detached cells and debris, serum-free medium was added into the 96-well plates and incubated under the usual culture conditions (37°C, 5% CO₂) for 48 h. Wound closure photographs were captured using a light microscope (DFC500, Leica Microsystems GmbH) at indicated time points and the outcomes were analyzed.

EMT was achieved when the 1×10⁴ HEp-2 cells were stimulated with EGF (30 ng/ml) for 48 h at 37°C. Subsequently, the cells were treated with genistein (20 μM) or genistein+TSA (20 μM: 100 nM), in the presence or absence of EGF, for another 48 h at 37°C. Alterations in cell morphology were documented by visualization using an inverted phase contrast microscope (DFC500; Leica Microsystems GmbH, Wetzlar, Germany).

The invasion assay was performed using Transwell plates (pore size, 8 µm) with a Boyden chamber (Sigma-Aldrich; Merck KGaA). Transfected cells were washed twice with serum-containing RPMI-1640 medium (Sigma-Aldrich; Merck KGaA). A total of 1×10⁵ T24 and SW780 cells were seeded onto the Transwell apparatus. Each insert was preloaded with Matrigel (50 µg; Sigma-Aldrich; Merck KGaA). Cells were suspended in 100 µl RPMI-1640 serum-free medium (Sigma-Aldrich; Merck KGaA) and placed in the top chambers. RPMI-1640 medium (100 µl; Sigma-Aldrich; Merck KGaA) containing 10% fetal calf serum (Sigma-Aldrich; Merck KGaA) was added to the bottom chambers. The chambers were incubated for 24 h at 37°C with 5% FBS in the lower chamber. Cells on the upper layer were removed by cotton buds and then washed with PBS. The cells that had invaded into the lower chambers were fixed with methanol for 15 min (Sigma-Aldrich; Merck KGaA) and stained with 5% crystal violet for 30 min at 20°C (Sigma-Aldrich; Merck KGaA). The results were visualized by light microscopy (DFC500; Leica Microsystems GmbH, Wetzlar, Germany) and the final values represent the mean from three fields on the membrane. The results were visualized at magnification, ×100. Experiments were performed in triplicate.