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. 2020 Nov 17;3:682. doi: 10.1038/s42003-020-01408-z

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© The Author(s) 2020

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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Fig. 9 — Immunohistochemistry and immunoblot based analyses showed that METH induces a marked reduction of phosphorylation of CREB at the Serine133 residue in both human and mouse hearts. a Left panel: representative micrographs of immunohistochemical detection of phosphorylated CREB at Ser133 (brown color, positive DAB staining) in human non-METH user (n = 4) and METH toxicity (n = 4) LV sections. Hematoxylin (blue) was used to counterstain nuclei. Right panel: the bar graph represents the fold change of DAB-positive pCREBSer133 staining intensity in the nucleus of METH toxicity human hearts compared with non-METH user human hearts. Scale bar: 50 µm. b Left panel: representative micrographs of immunohistochemical detection of phosphorylated CREB at Ser133 (brown, DAB staining) in the vehicle-treated (n = 4) and METH-treated (n = 4) mouse LV heart sections. Hematoxylin (blue) was used to counterstain the nuclei. Right panel: the bar graph represents the fold change of DAB-positive pCREBSer133 staining intensity in the nucleus of the hearts of METH-treated mice compared to vehicle-treated mice. Scale bar: 50 µm. c Immunoblot analysis of total CREB and phosphorylated CREB at Ser133 (pCREBSer133) in vehicle- and METH-treated mouse heart-whole-cell lysates. Ponceau S staining of the transfer membranes was used to confirm approximately equal loading and transfer across the gel. Bar graphs represent densitometric quantification of total CREB and pCREBSer133 expression in the hearts of vehicle- and METH-treated mice (n > 5 mice per group). Boxes depict interquartile ranges, lines represent medians, and whiskers represent ranges. P values were determined using a two-tailed unpaired Student’s t test. P<0.05 between groups was considered statistically significant. d Immunofluorescent micrographs of pCREBSer133 staining (green) in vehicle- and METH-treated (100 µM) neonatal rat cardiomyocytes (NRC) show marked reduction of pCREBSer133 in cardiac Troponin I (red)-positive cardiomyocytes. DAPI (blue) was used to counterstain the nuclei. Images are representative of three independent experiments. Scale bar: 20 µm. Veh vehicle, METH methamphetamine.