FIG. 5.

Gas6 does not act as a growth-potentiating factor for C57MG cells. (A) Gas6-induced proliferation is maintained in serum-starved C57MG cells. C57MG cells were plated at 10⁴ per cm² in 10% FCS–DMEM and were allowed to achieve DDI in the presence of serum for 3 days. Then cells were either maintained in the same medium (DDI), or the medium was changed to 0.5% FCS (DDI + 0.5%) and further incubated for 24 h. Serum-starved cells (48 h; 0.5%) were obtained by changing the medium to 0.5% FCS on the day after seeding and were incubated for 2 days. The ability to induce DNA synthesis was tested by adding to the culture medium 400 ng of Gas6/ml together with 50 μM BrdU for 20 h. An immunofluorescence assay was performed as described in Materials and Methods. Unt, untreated. (B) Analysis of Gas6-induced downstream signaling on serum-starved DDI C57MG cells. DDI + 0.5% cells were prepared and treated as described for panel A. When wortmannin (100 nM), PD98059 (20 μM), or SB203580 (20 μM) was used, each was added to the culture medium for 1 h before stimulation with Gas6 (400 ng/ml). Entry into the S phase was monitored after 20 h as described above. An analysis of Akt and MAPK activation was performed: Western blotting with antibodies specific to Thr308 of Akt (P-Akt) or raised against Thr202/Tyr204 of p44/42 MAPK (P-MAPK). C57MG cells were prepared as above, and 1 h before Gas6 stimulation, the medium was changed to serum-free DMEM. In separate experiments, cells were treated with wortmannin (100 nM), PD98059 (20 μM), or SB203580 (20 μM) before Gas6 stimulation. Parallel membranes were decorated with anti-Akt or anti-MAPK to monitor the total amount of kinase in the lysate. Cells were separately stimulated with 10% FCS and used as positive control (FCS). −, absence; +, presence.