Fig. 2. Abrogation of Smad4 promotes lung cancer cell migration and invasion.

a Primary lung cancer cells were isolated from lung tumors of PK and SPK mice and immortalized. The protein levels of Smad4 were examined in PK and SPK cells, and the experiment was repeated three times independently with similar results. Each lane represents a cell sample from an individual mouse. b The role of Smad4 in murine lung cancer cell migration/invasion was tested by Transwell assay and the experiment was repeated three times independently with similar results. Scale bar, 100 μm. c Smad4 inhibited wound healing in cell culture. Cells were made a wound between the two red dashed lines, the area of the two dashed lines represent the level of wound healing and the cell migration activity. Wound healing percentage is the ratio of the wound healing area and the primary wound area, and the experiment was repeated three times independently with similar results. Scale bar, 100 μm. d Transfection of Smad4 plasmid in SPK cells increased the protein level of Smad4, and the experiment was repeated three times independently with similar results. e Overexpression of Smad4 in SPK cells decreased cell migration/invasion, and the experiment was repeated three times independently with similar results. Scale bar, 100 μm. f The number of migrated cells was quantified. Data presented are the mean ± SD from three biological replicates (n = 3); ***p = 6.7544E-05; **p = 0.000211036; as determined by two-tailed Student’s t-test.