JCS03236 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Src activation in serum-starved 5637 cells is sensitive to PP2 and SU6656. 5637 cells were serum-starved for 24 hours in the absence or the presence of PP2 (10 μM), PP3 (10 μM), SU6656 (1 μM), K252a (10 μM), or U0126 (10 μM). After the treatments, whole cell extracts were prepared and analyzed for activation of Src as in Fig. 2.

• Supplemental Figure 2 -

  Fig. S2. Reversal of serum-starvation-induced tyrosine phosphorylation of p145 by addition of serum. Serum-starved 5637 cells (24 h) were treated for the indicated times with 10% FCS. After the treatments, whole cell extracts (30 μg/lane) were prepared and analyzed by immunoblotting with anti-phosphotyrosine antibody (PY99). The position of pp145 is indicated. The extract prepared from normally grown 5637 cells (FCS) was also analyzed as a control.

• Supplemental Figure 3 -

  Fig. S3. (A) Time-dependent activation of caspase-like protease in serum-starved 5637 cells. Whole cell extracts were prepared from 5637 cells that had been cultured for 0-24 hours without serum plus 10 μM PP2, and subjected to protease assay (10 μg/assay) toward Ac-DEVD-MCA as in Fig. 1D. (B) PP2 readily promotes activation of caspase-like protease in serum-starved 5637 cells. 5637 cells were cultured without serum for 24 hours. After the treatment, the cells were further treated with 10 μM PP2 for 0, 30 or 60 minutes in the serum-free medium. Protein tyrosine phosphorylation and protease activity toward Ac-DEVD-MCA were evaluated by anti-phosphotyrosine immunoblotting (IB: PY99) (30 μg/lane) and protease assay (10 μg/assay), respectively. Data obtained with normally cultured cells (+ FCS) were also shown. Data shown are representative of three independent experiments. (C) Indirect fluorescent analysis of nuclear morphology in 5637 cells. 5637 cells were cultured in 10% FCS for 48 hours and then in four different conditions (10% FSC plus 10 μM PP3, 10% FCS plus 10 μM PP2, no serum plus 10 μM PP3, or no serum plus 10 μM PP2) for additional 48 hours. After the treatments, the cells were subjected to indirect fluorescent staining of the nucleus and confocal laser scanning microscopic analysis as described in Materials and methods.

• Supplemental Figure 4 -

  Fig. S4. (A) CuCl₂ does not block tyrosine phosphorylation of p145^met in serum-starved 5637 cells. 5637 cells were treated in normal condition (10% FCS, control), HGF-containing normal conditions (125 ng/ml for 1 hour or 24 hours), or serum-free condition (- FCS, 24 h) in the absence or the presence of 1.25 mM CuCl₂ or MgCl₂. After the treatments, whole cell extracts were prepared and analyzed by immunoprecipitation and/or immunoblotting as in previous figures: direct immunoblotting was done with use of 20 μg/lane; immunoprecipitation was done with use of 200 μg/lane. The positions of pp145^met, p145 ^met, pp60^src, p60 ^src, pp44, and pp42 are indicated. (B) Growth rate of serum-starved 5637 cells is not affected by CuCl₂. 5637 cells (1×106 cells/dish) were cultured in normal condition (10% FCS) for 48 hours. After the treatment, cells were further cultured for additional 48 hours in normal condition (open circles), 10% FCS plus 1.25 mM CuCl₂ (open triangles), 10% FCS plus 125 ng/ml HGF (open squares), 10% FCS plus 125 ng/ml HGF plus 1.25 mM CuCl₂ (closed squares), without serum (closed circles), or without serum plus 1.25 mM CuCl₂ (closed triangles). Cell number was determined at 24, 48, 72,and 96 hours post initial treatment. (C) Caspase activity is not induced by CuCl₂ in serum-starved 5637 cells. 5637 cells were treated as in B. After the treatments (96 hours post treatment), cell death (black bar) and caspase 3/7 protease activity (grey bar) of the whole cell extracts (20 μg/assay) were determined. Data shown are representative of three independent experiments.