WM1650-TBX3 cells have increased anchorage independence and migratory ability. Cell growth (a) in a 1% agar slurry and visualised with p-iodonitrotetrazolium chloride stain and (b) in 0.8% methylcellulose and assessed by the MTT assay. Each data point represents the mean ± SD from at least three independent experiments (*p < 0.05; **p < 0.0009). (c,d) Migratory ability of the WM1650-TBX3 and WM1650-ctrl cells was compared using (c) in vitro scratch and (d) transwell motility assays. Each data point represents the mean ± SD from at least three independent experiments (*p < 0.05). (e) Real-time PCR analysis (left panel, each data point represents the mean ± SD from at least three independent experiments (*p < 0.05)) and western blotting (right panel; representative of three different experiments) of TBX3 and E-cadherin expression in WM1650-ctrl and WM1650-TBX3 cells. The expression of E-cadherin was quantified as the densitometry value (pooled from three different experiments) analysed by UN-SCAN-IT gel 6.1 software and was normalised to p38 levels as shown in the table below. (f) WM1650-TBX3 or WM1650-ctrl cells were injected subcutaneously into the flanks of 6 nude mice (3 males and 3 females for each cell line) and 29 days post-injection, mice were euthanized, photographed and tumours excised for histological analyses. (g) Representative tumour section stained with haematoxylin and eosin and photographed at 20× magnification. Black arrows show tumour cells invading skeletal muscle.