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. 2018 Aug 6;1:104. doi: 10.1038/s42003-018-0108-5

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© The Author(s) 2018

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. 6 — aPC reverses glucose-induced sustained p66^Shc expression and the pro-atherogenic phenotype of macrophages. a, b Representative immunoblot images (out of four independent repeat experiments with two technical replicates each, left) and dot plot summarizing relative expression levels (right panels) of p66^Shc, CD36, and GAPDH (loading control) in mouse bone marrow-derived macrophages (BMDMs) exposed to different lipid (a) or glucose (b) concentrations without or with additional aPC treatment. c Representative immunoblot images (out of 6 independent repeat experiments with two technical replicates each, top) and dot plot summarizing data (bottom) of p66^Shc and GAPDH (loading control) in BMDMs isolated from non-hyperglycaemic (control, Cont), hyperglycaemic (DM, 22 weeks persistent hyperglycaemia), transiently hyperglycaemic (DM-NG, 16 weeks of sustained hyperglycaemia followed by 6 weeks of SGLT2-inhbitor treatment), or DM-NG-aPC (DM-NG mice with aPC treatment parallel to SGLT2-inhibitors treatment). d Representative immunoblot images of p66^Shc (out of four independent repeat experiments with two technical replicates each) and GAPDH (loading control) in wild-type (WT), PAR1^−/−, PAR2^−/−, or PAR3^−/− BMDMs (d, left panel) and in WT-BMDMs exposed to inhibitory antibodies of PAR4 or EPCR (d, right panels). NL: normal LDL; HL: high LDL (50 µg/ml, 48 h); HL-NL: 50 µg/ml LDL, 48 h, followed by NL, 24 h); NG: normal glucose (5 mM glucose plus 20 mM mannitol, NG, 48 h); HG: high glucose (25 mM, 48 h); HG-NG: high glucose followed by normal glucose (25 mM glucose, 48 h, followed by 5 mM glucose, 24 h), HG-NG-aPC: HG-NG treated in addition with aPC (20 nM for the last 24 h); Data shown as dot plots represent mean ± SEM of at least 3 independent repeat experiments with at least two technical replicates each; **P < 0.01; one-way ANOVA with Bonferroni-adjusted post hoc comparison of HL versus NL and HL-NL versus HL (a), HG and HG-NG versus NG and HG-NG-aPC versus HG-NG (b, d), DM and DM-NG versus Cont and DM-NG-aPC versus DM-NG (c), HG-NG-aPC (αPAR4 and αEPCR-4 BMDMs) versus HG-NG-aPC (d, right panel) BMDMs. One-way ANOVA with Bonferroni-adjusted post hoc comparison of WT (HG and HG-NG versus NG and HG-NG-aPC versus HG-NG) and HG-NG-aPC (PAR3^−/− and PAR2^−/− BMDMs) versus HG-NG-aPC (PAR1^−/− BMDMs) (d, left panel). Uncropped immunoblots for Fig. 6a–d are provided in Supplementary Figure 20 and Supplementary Figure 21, respectively