Figure 6

Cell survival by IGFBP5 is not affected by IGF1 or mediated by IGF1R. (A) LX2 cells were transfected with IGFBP5 small interfering RNA (siBP5). Sixteen hours after transfection the medium was replaced by serum-free medium for 48 h and then the viability was determined by performing a WST assay. Recombinant IGFBP5 (rBP5), IGF1 (rIGF1), or both (rIGF1+ rBP5), were added at 24 hand 45 h. (B) To cells treated as above, but cultured in 10% fetal calf serum, 0.5 μM gliotoxin was added at 45 h followed by a Caspase 3/7 assay 3 h later. (C) LX2 cells were cultured in serum-free medium for 48 h. rIGF1 was added in different concentrations at 24 h and 45 h, bromo-2'-deoxy-uridine (BrdU) was added at 32 h. Cells were lysed 16 h later (at 48 h) and assayed for BrdU incorporation. (D) LX2 cells were transfected with control (siCtrl) or siRNA for IGF1R (siIGF1R). Sixteen hours after transfection the medium was replaced by serum-free medium and cells were cultured for additional 48 h. The cells were then lysed and used for Western blot analysis in order to detect IGF1R. (E) Cells were transfected and cultured as in D. rIGF1 or rIGFBP5 (rBP5) was added at t = 24 h and 45 h after replacing medium. After 48 h a WST assay was performed. (F) 0.5 μM gliotoxin was added at 45 h, after replacing the medium. Caspase activity was measured 3 h later. All results are given as a percentage control cells.