WM1650-TBX3 cells have decreased proliferative ability. (a) Real-time PCR (each data point represents the mean ± SD from at least three independent experiments (*p < 0.05)) and (b) western blot (representative of three different experiments) analyses of TBX3 mRNA and protein levels respectively in WM1650-ctrl and WM1650-TBX3 cells. p38 was used as a loading control. (c) Growth curve assays of cells grown in medium supplemented with 10% or 2% FBS. Each data point represents the mean ± SD from at least three independent experiments (*p < 0.05) (d) 5-bromo-2-deoxyuridine (BrdU) incorporation assay. Graph shows an average of BrdU-positive cells expressed as a percentage of total cells counted at 1 hr and 8 hrs in 20 fields of view (*p < 0.05, mean +/− SD). (e) Cell cycle distribution was determined by staining cells with propidium iodide and measuring their DNA content by flow cytometry. Percentage of cells in each phase of the cell cycle are shown in the graph below (*p < 0.05, mean +/− SD). (f) Western blotting (representative of three different experiments) of the indicated proteins in WM1650-ctrl and WM1650-TBX3 cells. The expression of TBX2, p53, p21, p16 and p14 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 tables below.