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. 2020 Jan 21;10:842. doi: 10.1038/s41598-020-57673-9

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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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BH4 was key factor of CRIF1 deficiency-mediated eNOS uncoupling in HUVECs. (A) Quantified DCF-DA fluorescence of endothelial cells with different BH4 concentration and presence or absence of CRIF1 siRNA (n = 3 per group; ^*P < 0.05 vs con; ^#P < 0.05 vs CRIF1 siRNA 100 pmol). (B) Nitrite and nitrate measurement in supernatant media from control and CRIF1 siRNA treated HUVECs with or without BH4 (10 µM) and L-Arg (10 mM) (n = 3 per group; ^*P < 0.05 vs control; ^#P < 0.05 vs CRIF1 siRNA 100 pmol). (C) Nitrite and nitrate measurement in supernatant media from cells with different concentration of BH4 and with or without CRIF1 siRNA (n = 3 per group; *P < 0.05 vs. control; ^#P < 0.05 vs CRIF1 siRNA 100 pmol). (D,E) Phosphorylation at serine 473 of Akt and phosphorylation at serine 1117 of eNOS in CRIF1-downregulated HUVECs with different amount of BH₄. Measurement of band density was used to quantify protein expression (n = 3 per group; *P < 0.05 vs. control; ^#P < 0.05 vs CRIF1 siRNA 100 pmol).