Research Paper Volume 15, Issue 1 pp 164—178

Figure 4. Dox-induced ROS production, cell apoptosis, downregulation of pro-survival signaling, and upregulation of IGFBP3 expression are dependent upon HIF1A activity. (A) H9c2 cells were incubated with increasing dose of Dox for 24 h. HIF1A protein levels were analyzed in H9c2 cells and animal cardiac tissue using western blotting. (B) Nuclear translocation of HIF1A in cells challenged with or without Dox were detected using fluorescence microscopy. Blue represents nucleus; green represents HIF1A. (C, D) Dox-exposed cells were treated with HIF1A inhibitor (C) or transfected with Hif1a siRNA (D). The levels of IGFBP3, pro-survival, and pro-apoptotic proteins in cells and cell culture medium (for secreted IGFBP3) were detected using western blotting. (E, F) Dox challenged H9c2 cells either treated with HIF1A inhibitor or transfected with siHif1a, were incubated with TUNEL and MitoSOX reagents to assess apoptosis mediated cell death (E) and mitochondrial ROS production (F). Statistical significance difference was shown as ^*P < 0.05, ^**P < 0.01 from at least three independent experiments.