Figure 2

HoxD10 inhibits proliferation, and impairs cell migration and invasion of gastric cancer cells. (A) Overexpression of HoxD10 in AGS gastric cancer cells was confirmed by RT-PCR (left lane) and Western blot (right lane). GAPDH and β-actin were used as internal controls. (B) The monolayer colony formation assays. AGS cells were transfected with pcDNA3.1-HoxD10or pcDNA3.1 empty vector and selected with G418 for 14 to16 d. The numbers of survival colonies in each empty vector-transfected control were set to 100%, while HoxD10-transfected cells were presented as mean percentage ± SD from three individual experiments in right diagram. (C) The number of viability cells was measured by MTS cell proliferation assays. The assay was performed at 6, 24, 72 or 120 h in AGS cells transfected with pcDNA3.1-HoxD10or pcDNA3.1 empty vector. Experiments were performed in triplicate. The asterisk indicates statistical significance (*P < 0.05, **P < 0.01). (D) Cell migration assays were performed in modified Boyden transwell chambers assay. 5 × 10⁴ AGS cells/well were plated and incubated for 24 h. The migratory cells on the bottom of the membrane were fixed and nucleus stained with DAPI (upper figures). The mean number of visible cells was counted by fluorescence microscope in five random high power fields (lower bar diagram). (E) QC 24-Well Collagen-Based Cell Invasion Assay (Millipore, USA) was used to assess cell invasion. Invaded cells were stained with Cell Stain Solution, then detected on a standard microplate reader (560 nm). The asterisk indicates statistical significance (*P < 0.05, **P < 0.01). B, E: ■, Vector; □, HoxD10; C: —?—, vector; —■—, HoxD10.