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Fig. S1. Src-dependent proliferation of human bladder carcinoma cells under serum-starved culture conditions. Cells (1×105 cells per dish) were cultured for 48 h in normal media (serum +) (black bars: cell number at this point is employed as 100%) and then cultured further for additional 48 h under serum-starved conditions in the absence (serum −) (grey bars) or the presence of 10 µM PP2 (serum −, PP2 +) (dot-patterned bars). After the treatments, cell number was determined. Data shown are mean + standard deviations of three or four independent experiments. *P<0.01 compared with control data. **P<0.01 compared with data for serum −.
Fig. S2. MAPK, but not Src, contributes to the survival and proliferation of human bladder carcinoma cells in serum-containing culture conditions. (A) Six carcinoma cells used in Fig. 1 were cultured under serum-starved conditions for 24 h (serum −) and stimulated with 10% FCS for 10 min (serum +). Triton X-100-solubilized cell extracts were prepared and analyzed for the expression and/or phosphorylation of Src and MAPK as in Fig. 1A. (B) Shown is the summary of the serum-dependent activation of Src and MAPK, and the effect of PP2 or U0126 on the serum-dependent proliferation of human carcinoma or immortalized cells as used in Fig. 1B. Note that only statistically significant values were taken as those showing “Src or MAPK is activated”.
Fig. S3. Identification of 45-kDa UPIIIa in 5637 cells. Triton X-100-solubilized cell extracts were prepared from Xenopus unfertilized eggs (30 µg/lane) and 5637 cells (20 µg/lane), and analyzed by immunoblotting with either a specific antibody against the extracellular domain of Xenopus UPIII (IB: UPIII) or a control preimmune antibody (IB: preimm.). Black and grey arrowheads indicate the positions of a 45-kDa UPIIIa in 5637 cells and a 30-kDa UPIII in Xenopus eggs, respectively.
Fig. S4. Effect of a synthetic protease inhibitor peptide on partial proteolysis of UPIIIa in serum-starved 5637 cells. 5637 cells were serum-starved in the absence or the presence of a synthetic peptide (UPIII-GRR, UPIII-CT, or BOC-GRR-MCA: at 100 µM) for 24 h. Triton X-100-solubilized cell extracts were prepared and analyzed for tyrosine phosphorylation of Met (upper panel, an arrowhead indicates the position of the tyrosine-phosphorylated Met) and the partial proteolysis of UPIIIa (lower panel, the positions of intact and fragmented UPIIIa are indicated by a closed and opened arrowheads, respectively) as in Fig. 2A and Fig. 4C, respectively. Data obtained with the normally cultured cells (FCS +) were also shown.
Fig. S5. Effect of PP2 and genistein on partial proteolysis of UPIIIa in serum-starved 5637 cells. 5637 cells were serum-starved in the absence or the presence of tyrosine kinase inhibitors or their inactive analogs (genistein or daidzein at 50 µM, PP2 or PP3 at 5 µM) for 24 h. Triton X-100-solubilized cell extracts were prepared and analyzed for tyrosine phosphorylation of Met (upper panel, an arrowhead indicates the position of the tyrosine-phosphorylated Met) and the partial proteolysis of UPIIIa (lower panel, the positions of intact and fragmented UPIIIa are indicated by a closed and opened arrowheads, respectively) as in Fig. 2A and Fig. 4C, respectively.
Fig. S6. List of genes showing Src-dependent up-regulation in serum-starved 5637 cells. Shown is the list for sixty-six genes (except for HB-EGF), whose expression were up-regulated more than 2-fold under serum-starved conditions and down-regulated more than 2-fold in serum-starved, PP2-containing conditions, as described in Fig. 7C.
Fig. S7. List of genes showing Src-dependent down-regulation in serum-starved 5637 cells. Shown is the list for forty-five genes, whose expression were down-regulated more than 2-fold under serum-starved conditions and up-regulated more than 2-fold in serum-starved, PP2-containing conditions, as described in Fig. 7C.
