IGFBP5 impact on apoptosis in LX2 cells. (A) LX2 cells were cultured in serum-free medium for 48 h. Recombinant IGFBP5 (rBP5) was added at 24 h and 45 h and, at 48 h, a Caspase 3/7 assay was performed. (B) Cells grown in 10% fetal calf serum were treated with rBP5 as above, with the addition of 0.5 μM gliotoxin after 45 h. (C) LX2 cells were transfected with small interfering control (siCtrl) or IGFBP5 siRNA (siBP5). Sixteen hours after transfection, the medium was replaced by serum-free medium and cells were cultured for additional 48 h before performing the caspase assay. To siIGFBP5 transfected cells, recombinant IGFBP5 was added at t = 24 h and 45 h (siBP5+rBP5). (D) To cells treated with si- and r-BP5 as in C, 0.5 μM gliotoxin was added 3 h before performing the caspase assay. All results are shown as a percentage of the control cells. (E) Apoptosis was induced in LX2 cells transfected with control siRNA (siCtrl) or IGFBP5 siRNA (siBP5) by the addition of 0.5 uM gliotoxin at t = 45 h. Proteins were isolated at 48 h and Western blotting was performed for poly(ASP-ribose) polymerase detection (left panel). Quantification is shown in the right panel. β-actin was used as a loading control.