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. 2017 Oct 18;7:13424. doi: 10.1038/s41598-017-13949-1

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Effects of P-BBG diet on levels of myocardial oxidative stress, cleaved caspase-3, HIF-1α, pAkt/Akt, pSTAT3/STAT3 and pNF-kB/NF-kB. (A) Representative images of DHE (dihydroethidium) staining of Control and P-BBG left ventricles. (B) P-BBG diet (n = 15) prevented an increase in anion superoxide generation following cardiac ischemia/reperfusion injury compared to Control diet (n = 15); the results are shown as arbitrary units of fluorescence intensity (see Methods). (C) The myocardial levels of cleaved caspase-3 were lower in P-BBG (n = 7) than Control (n = 7) hearts. Levels of cleaved caspase-3 are expressed as arbitrary units of cleaved caspase-3 (19 kDa, MW)/GAPDH (37 kDa, MW) ratio. Representative images of cropped densitometric bands of cleaved caspase 3 AND GAPDH are shown. The full-length blots/gels are presented in Supplementary Figure 1C. (D) The myocardial levels of HIF-1α are similar in P-BBG (n = 7) and Control (n = 7) hearts. Levels of HIF-1α are expressed as arbitrary units of HIF-1α (110 kDa, MW)/GAPDH (37 kDa, MW) ratio. Representative images of cropped densitometric bands of HIF-1α and GAPDH are shown. The full-length blots/gels are presented in Supplementary Figure 1C. (E) The myocardial levels of p-Akt/Akt ratio are similar in P-BBG (n = 7) and Control (n = 7) hearts. Levels of p-Akt (60 kDa, MW)/Akt (60 kDa, MW), normalized to GAPDH levels, are expressed as arbitrary units. Representative images of cropped densitometric bands of p-Akt, Akt and GAPDH are shown. The full-length blots/gels are shown in Supplementary Figure 1C. (F) The myocardial levels of p-NF-kB/NF-kB ratio are similar in P-BBG (n = 7) and Control (n = 7) hearts. Levels of p-NF-kB (65 kDa, MW)/NF-kB (65 kDa, MW), normalized on GAPDH levels, are expressed as arbitrary units. Representative images of cropped densitometric bands of p-NF-kB, NF-kB and GAPDH are shown. The full-length blots/gels are presented in Supplementary Figure 1D. (G) The myocardial levels of p-STAT3/STAT3 ratio are similar in P-BBG (n = 7) and Control (n = 7) hearts. Levels of p-STAT3 (86 kDa, MW)/STAT3 (86 kDa, MW), normalized on GAPDH levels, are expressed as arbitrary units. Representative images of cropped densitometric bands of p-STAT3, STAT3 and GAPDH are shown. The full-length blots/gels are presented in Supplementary Figure 1E. P-BBG: low fat diet supplemented with barley beta-D-glucan enriched pasta (3 g/100 g of dry weight); HIF-1α: hypoxia-inducible factor 1-alpha; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; p-Akt: phospho-Akt; p-NF-kB: phospho- nuclear factor Kappa b; p-STAT3: phospho- signal transducer and activator of transcription 3. All measurements are mean ± SD. *p < 0.05 vs. Control.