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. 2013 Jun 12;169(5):1058–1071. doi: 10.1111/bph.12185

Figure 3.

Figure 3

TNF-α is involved in OGD/R-mediated apoptotic death. STS inhibits OGD/R-induced NF-κB activation and subsequent TNF-α expression to protect cardiomyocytes against apoptosis. Cells were subjected to 6 h OGD and 18 h recovery with or without the following indicated treatments: (A) Total protein (30 μg) or cytoplasmic or nuclear extracts (15 μg) were analysed by Western blot with antibodies specific for the indicated proteins, with β-actin as a total protein-loading control, Histone H3 (nuclear marker) and β-actin (cytoplasmic marker) as loading controls for nuclear and cytoplasmic extracts respectively. Results are means of three independent experiments. (B) Cytoplasmic extracts were analysed by Western blot with anti-p-NF-κB p65 and β-actin antibodies. Results are means of three independent experiments. (C) The specific DNA-binding activity of nuclear NF-κB was analysed with EMSA using a probe corresponding to the NF-κB binding site. Specificity of binding was confirmed by cold competition experiments with 50-fold molar excess of unlabelled NF-κB duplex oligonucleotide. The arrow indicates the NF-κB-DNA complexes. The graph shows results from one out of three independent experiments. (D) Cells were subjected to 6 h OGD and 4 h recovery with or without STS. Cytoplasmic extracts were analysed by Western blot with antibodies specific for the indicated proteins. Results are means of three independent experiments. (E and G) TNF-α and β-actin mRNA expression levels were determined using RT-PCR. Results shown are means of five independent experiments. (F and H) TNF-α release was measured using elisa. Results are expressed as mean values ± SD of three independent experiments performed in triplicate. ###P < 0.001, significantly different from control, **P < 0.01, **P < 0.001, significantly different from OGD/R alone. (I and J) Apoptosis of cells was determined with Annexin V-FITC-based flow cytometry. The annexin V-FITC +/PI- population of gated cells was measured with CellQuest software, and is presented as the apoptosis ratio. Results are expressed as means ± SD of three independent experiments performed in triplicate. ###P < 0.001, significantly different from control, ***P < 0.001, significantly different from OGD/R alone. (K and L) Total protein (30 μg) was analysed by Western blot with antibodies specific for cleaved caspase-3 and caspase-3. β-actin was used as the loading control. Data are means of three independent experiments.