Research Paper Advance Articles pp 24345—24356

Implication of integrins α3β1 and α5β1 in invasion and anoikis of SK-Mel-147 human melanoma cells: non-canonical functions of protein kinase Akt

Figure 1. Downregulation of the expression of α3β1 and α5β1 integrins reduces clonal activity and enhances anoikis of SK-Mel-147 cells. The cells were transduced with a lentiviral plasmid vector pLKO.1-puro containing α3- or α5- shRNA or with the control (scrambled shRNA) vector and selected using puromycin. (A) Western-blotting of the cellular lysate proteins. Cell lysate proteins were run on SDS-PAGE, western-blotted and probed as described in Materials and Methods. The blots were probed with 1:1000 dilution of antibodies to the specified proteins and treated as described in Materials and Methods. Shown are representative blots. Numbers below the bands indicate the ratio (%) protein level in integrin shRNA transfected cells compared to control shRNA transfected cells normalized against β-actin. Results of three independent experiments are shown (M ± SEM). *ρ < 0.05, **non-significant. (B) Effect of α3β1 or α5β1 downregulation on clonal capacity of the cells. Cells transduced with the scrambled or α3/α5- shRNA-containing vectors were treated as described in Materials and Methods; results of three independent experiments are shown (M ± SEM); *p < 0.01, relative to control. (C) Effect of the downregulation of α3β1 or α5β1 on anoikis of the cells. Cells were transduced with the scrambled (PLKO.1) or α3- or α5- shRNA-containing vectors and cultured on poly-HEMA as described in Materials and Methods.