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PLC-1 is required for IGF-I protection from cell death induced by loss of extracellular matrix adhesion

¹ Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee, TN 37232-0146, USA
² Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee, TN 37232-0146, USA

View larger version (11K): [in a new window]   Fig. 1. The influence of PLC-1 on cell survival. (A) Null and Null+ cells were plated on either regular tissue culture plastic dishes to adhere or on tissue culture dishes coated with poly-HEMA for suspension culture. 10% FBS was added as indicated (+ Serum) and cell viability was measured 20 hours later by calculating the ratio of trypan-blue-positive cells to live cells. The open and closed bars represent Null and Null+ cells, respectively. Each bar indicates the average of triplicate assays. SF indicates serum-free media. (B) Null and Null+ cells were seeded on poly-HEMA coated dishes. 10% FBS or a broad spectrum caspase inhibitor Z-VAD-FMK (20 µM) were added as indicated. Cells were collected 20 hours later, washed with PBS and cell lysates were prepared using cell lysis buffer. Caspase 3 activity present in the cell lysates was determined as described in the Materials and Methods. Open and closed bars indicate Null and Null+ cells, respectively.

View larger version (15K): [in a new window]   Fig. 2. Capacity of IGF-I to protect Null and Null+ cells from loss of extracellular-matrix-induced cell death. Both TVI (A,C) and TVII (B,D) Null and Null+ cells were placed in suspension by seeding on poly-HEMA coated dishes. IGF-I (100 ng/ml) was added to the medium as indicated, and the cells were harvested 20 hours later. Cell viability was determined using trypan blue staining (A,B) or caspase 3 activity (C,D). Open and closed bars represent results for Null and Null+ cells, respectively.

View larger version (53K): [in a new window]   Fig. 3. The level of IGF-I receptor and its autuphosphorylation in Null and Null+ cells. Adherent Null and Null+ cells were incubated at 37°C in medium containing 10% FBS. At a confluence level of 50-70%, the cells were serum-starved by incubating overnight in medium containing 0.5% FBS. After trypsinization the cells were placed in suspension culture by plating on poly-HEMA coated dishes. IGF-I (100 ng/ml) was added as indicated and incubated at 37°C for 15 minutes. The cells were then lysed in TGH buffer, and the cell extracts were subjected to immunoprecipitation. (A) An aliquot (1 mg) of lysate was immunoprecipitated with IGF-I receptor antibody and analyzed by western blotting with anti-phosphotyrosine. (B) The phosphotyrosine blot was stripped and reprobed with IGF-I receptor antibody.

View larger version (12K): [in a new window]   Fig. 4. Capacity of ionomycin and TPA to rescue IGF-I-mediated survival in Null cells. Null cells were placed in suspension culture as described in Fig. 2. 10% FBS, IGF-I (100 ng/ml), or ionomycin (2 µM) and TPA (100 ng/ml) were added as indicated. Caspase 3 activity was measured 20 hours later.

View larger version (46K): [in a new window]   Fig. 5. Detection of IGF-I-dependent tyrosine phosphorylation. Null+ cells were either untreated or treated with 100 µM pervanadate (pV) for 15 minutes at 37°C. Then the cells were incubated with IGF-I (100 ng/ml) at 4°C for 45 minutes in suspension culture or in adherent condition as indicated. The cells were lysed in TGH buffer, and the cell extracts were subjected to immunoprecipitation. (A) An aliquot (1 mg) of lysate was immunoprecipitated with PLC-1 antibody and analyzed by western blotting with antiphosphotyrosine. (B) The phosphotyrosine blot was stripped and re-probed with PLC-1 antibody.

View larger version (11K): [in a new window]   Fig. 6. Pharmacological evaluation of signaling pathways that mediate the IGF-I-dependent cell survival of Null+ cells. Null+ cells were placed in suspension and IGF-I (100 ng/ml), LY294002 (50 µM), or PD 98509 (50 µM) were added as indicated. The cells were then incubated at 37°C for 20 hours, lysed, and caspase 3 activity present in the lysates was measured.