Figure 5
Role of HSP27 in Ntn-1-induced cytoprotective effects. (a) Cells were treated with Ntn-1 (10 ng/ml) for 30 min before hypoxia exposure for 72 h, and then HSP27, HSP60, HSP70, and HSP90 were detected by western blot. (b and c) Cells were transfected for 24 h with either HSF-1- or HSP27-specific siRNA (100 nmol/l) or non-targeting control siRNA (100 nmol/l) using Hyperfectamine prior to hypoxia with Ntn-1 exposure for 72 h. HSP27, HSP70, phospho-Aktthr308, phospho-Aktser473, and Akt expressions were analyzed using western blot. (d) Cells were pretreated with Ntn-1 (10 ng/ml) and/or Akt inhibitor (10 μM) for 30 min before being exposed to hypoxia, and Bax, COX IV, and β-actin in the cytosolic and mitochondrial fraction were detected by western blot. (e) Acquisition of JC-1 fluorescence images of mitochondria was performed using confocal microscopy. Quantification of mitochondrial membrane potential is expressed as a ratio of J-aggregate to JC-1 monomer fluorescence intensity. Values are expressed as the mean±S.E. of four independent experiments with triplicate measurement in one sample. *P<0.05 versus control, **P<0.05 versus hypoxia alone, #P<0.05 versus combination of hypoxia and Ntn-1. (f) Cells were pretreated with HSP27-specific siRNA or non-targeting control siRNA for 24 h prior to hypoxia with Ntn-1 exposure for 72 h and then harvested. Cyt c, COX IV, and β-actin in the cytosolic and mitochondrial fraction were detected by western blot. (g) Cells were exposed to hypoxia for 72 h and then immunoprecipitation of anti-cyt c was analyzed by western blotting with antibodies that recognize HSP27 or HSP70. (h) Cells were pretreated with HSP27-specific siRNA or non-targeting control siRNA for 24 h prior to hypoxia, with Ntn-1 exposure for 72 h and then harvested. Cell lysates were analyzed by western blotting with antibody to cyt c. Anti-HSP27 immunoprecipitation was analyzed by western blotting with cyt c antibody. Each of the examples (a–d and f–g) is representative of five independent experiments. The right part (a–d and f–g) depicting the bars denotes the mean±S.E. of five independent experiments for each condition determined from densitometry relative to β-actin or total Akt. *P<0.05 versus control, **P<0.05 versus hypoxia alone, #P<0.05 versus combination of hypoxia and Ntn-1. ROD, Relative Optical Density
