Figure 7.

GM-CSF—a specific mediator of cellular transformation in a paracrine manner.A, wound healing assay of NIH3T3 cells incubated with conditioned media (CM) collected from RAW 264.7-PELP1 overexpression clones along with 0% serum media and GM-CSF pure protein as controls. The left panel depicts percentage of wound closure, and the right panel consists of representative images of wound closure. B, transwell migration assay of NIH3T3 (in the upper chamber) incubated with CM collected from RAW 264.7-PELP1 overexpression clones (in the bottom chamber). The left panel depicts the number of migrated cells, and the right panel shows representative images of migrated NIH3T3 cells captured by phase contrast microscope connected to a camera. C, ELISA to confirm increase in expression of GM-CSF along with increase in PELP1 expression in RAW 264.7 cells. Increasing concentration of PELP1 expression plasmid was transfected in RAW 264.7 cells, and the CM collected were concentrated to measure the CSF-2 concentration. Western blot showed the increased expression of PELP1 in RAW 264.7 cells transfected with pcDNA vector and increasing concentration of PELP1 expression plasmid. D, ELISA to confirm GM-CSF depletion in RAW 264.7-PELP1 clones. The depletion was achieved by incubating CM with anti GM-CSF antibody for 1 h at 37 °C followed by adding protein A/G beads for 2 to 4 h at 4 °C. GM-CSF, granulocyte–macrophage colony-stimulating factor; PELP1, proline, glutamic acid, and leucine-rich protein.