Figure 6
Tyrosine mutants of Sprouty2 inhibit its tumor suppressive function. Stable transfectants of A549 over expressing either Y55F or Y227F mutant of Sprouty2 designated A549-Y55FSpr and A549-Y227FSpr respectively were created and assayed for their functional properties. (A) and (B) Colony formation assay: A549, A549-Y55FSpr and A549-Y227FSpr cells were cultured in soft agar to assess their anchorage-independent colony formation ability. (A) The colonies were counted and photographed on day 12. (B) Graphical representation of the number of colonies formed by each cell line. (C) Equal numbers (1 × 105 cells/well) of A549, A549-Y55FSpr and A549-Y227FSpr cells were allowed to proliferate for four days. Schematic representation of their proliferation rate represented by live cell count every 24 h. (D) In vivo tumor formation: A549, A549-Spr or A549-Y55FSpr or A549-Y227FSpr cells were injected into the subcutaneous tissue of SCID mice. Growth rate of tumors formed by the respective cell lines in SCID mice is represented as increase in tumor volume monitored for up to 5 weeks. (E) Migration assay: Cells 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 = 0.0029). (F) Western blot analysis of the cell lysates of A549, A549-Spr, A549-Y55FSpr and A549-Y227FSpr to check for the levels of phospho ERK p44/42 and total ERK p44/42.