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Blood, Vol. 107, Issue 2, 669-678, January 15, 2006
IGF-1 receptor tyrosine kinase inhibition by the cyclolignan PPP induces G2/M-phase accumulation and apoptosis in multiple myeloma cells
Blood Strömberg et al.
107: 669
Supplemental materials for: Strömberg et al
Files in this Data Supplement:
- Figure S1 (JPG, 27.5 KB)
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A. RPMI 8226 cells were stimulated for 5 min with 6.7 nM IGF-1. Cell lysates were prepared in 1% Triton X-100, 10% glycerol, 20 mM. HEPES pH 7.4, 150 mM NaCl and 5 mM EDTA lysis buffer. Immunoprecipitation was performed with 2 µg IGF-1R ab C-20 (Santa Cruz Biotechnology) and Protein G-Sepharose (Amersham) o/n at 4°C. Immunoprecipitates were subjected to electrophoresis on a 4-12% Bis-Tris gel (Novex). Detection was performed using p-Tyr (py99) or C-20 as described. B. RPMI 8226 cells were incubated with indicated concentrations of PPP for 30 min. Cell lysats of RPMI 8226 were prepared in mammalian extraction reagent (Pierce, Rockford, IL). Precipitation was performed using Seize columns (Pierce) packed with 20 µl IGF-1R ab (C20, Santa Cruz Biotechnology) and 25 µl sepharose, 50 % Protein G-Sepharose (Amersham). Detection was performed with p-Tyr (py99) and H60 as described.
- Figure S2 (JPG, 39.8 KB)
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RPMI 8226 cells were serum-starved o/n, treated with 1 µM PPP for 3 h and stimulated for 5 min with 6.7 nM IGF-1 or 100 nM Insulin. Cell lysates were prepared in 1% Triton X-100, 10% glycerol, 20 mM HEPES pH 7.4, 150 mM NaCl and 5 mM EDTA buffer. Immunoprecipitation was performed with 2 µg IRS-1 antibody (Santa Cruz Biotechnology) and Protein G-Sepharose (Amersham Biosciences) o/n at 4°C. The immunoprecipitates were subjected to electrophoresis on a NuPAGE gel. Analysis by Western blotting was performed as described using p-Tyr (PY-99) (Santa Cruz). Actin was used as loading control.
- Figure S3 (JPG, 37 KB)
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The R- and P6 mouse cell lines were from Dr. Baserga (Thomas Jefferson University, Philadelphia, PA). The R- fibroblasts are IGF-1R negative, and are derived from an IGF-1R knockout mouse embryo1. The P6 line is a 3T3 derivative overexpressing the human IGF-1R1. The cells were cultured in DMEM supplemented with 10% (R-) or 5% (P6) FBS. P6 and R- cell lines were cultured in the presence of G-418 (Promega). Cell viability determinations were performed using the Cell proliferation kit II (Roche Inc, Indianapolis, IN) based on colorimetric change of the yellow tetrazolium salt XTT in orange formazan dye by the respiratory chain of viable cells2.
1. Roehm, N.W., Rodgers, G.H. et al. (1991). An improved colorimetric assay for cell proliferation and viability utilizing the tetrazolium salt XTT. J Immunol Methods 142 (2):257-65.
2. Rubini, M., Hongo, A. et al. (1997). The IGF-I receptor in mitogenesis and transformation of mouse embryo cells: role of receptor number. Exp Cell Res 230(2): 284-92
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