Figure 1

Induction of Sprouty2 and inhibition of cell migration in JSRV Env transformed cells. (A) A549 cells were transfected with a plasmid carrying JSRV Env gene (T) or the empty vector (C), and the expression of Sprouty2 and β-actin was monitored on days 3 and 6 post transfection by RT-PCR using different template concentrations (500 ng and 250 ng). Data represent two independent experiments. (B) and (C) Quantitative RT-PCR analysis depicting the fold increase in Sprouty2 mRNA levels in the stable cell lines A549-Spr and A549-Env compared to A549 (*P < 0.0007, **P < 0.0001) (B) and in BEAS-2B-Env compared to BEAS-2B (C) *P < 0.0001. (D) Western blot analysis of the expression of Sprouty2 protein and β-actin in stably transformed cell lines compared to the parental cell lines. Data represent five independent experiments. (E) and (F) Cell migration assay: Equal number of A549, A549-Spr and A549-Env cells (E) or BEAS-2B and BEAS-2B-Env cells (F) were allowed to migrate across the 8 μm porous membrane in a Boyden chamber in response to serum. After 15 h, the migrated cells were fixed, stained and counted (*P < 0.0001). (G) The effect of 200 pmoles of Sprouty2-specific siRNA on the migration ability of A549, A549-Env, BEAS-2B and BEAS-2B-Env cells compared to control siRNA (*P = 0.014, **P = 0.0169, ***P = 0.034, ****P = 0.0111). Corresponding changes in Sprouty2 protein levels compared to β-actin control are depicted in the Western blot shown below the bar diagram for the respective cell lines. (C) indicates control siRNA and (Sp) indicates Sprouty2 siRNA. Data represent two independent experiments.