Research Paper Volume 15, Issue 1 pp 164—178

Figure 3. Doxorubicin enhances extracellular binding of IGFBP3 to IGF1, blocking IGFI survival signaling and promoting cell apoptosis. H9c2 cells were treated with 1.0 μM Doxorubicin (Dox) for 24 h. (A) Extracellular association of IGF1 with IGFBP3 was detected using co-IP. (B, C) Dox-challenged cells were incubated with either (B) anti-IGFBP3 antibody at indicated amounts or transfected with (C) Igfbp3 siRNA. The levels of pro-survival and apoptosis-related proteins were analyzed using western blotting. (D, E) H9c2 cardiomyoblasts challenged with Dox (1.0 μM) for 24 h were either incubated with anti-IGFBP3 antibody and/or transfected with Si-Igfbp3 and thereafter probed with TUNEL and MitoSOX reagents to evaluate the apoptotic cell death (D) and mitochondrial superoxide generation (E). Data are expressed as mean ± standard deviation (n = 3). Scale bar represents 100 μm. Statistical significance is showed as follows: ^*P < 0.05, ^**P < 0.01.