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. 2018 Mar 29;9:1281. doi: 10.1038/s41467-018-03668-0

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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. 5 — Role of eNOS during mesendodermal commitment. a Fluorescence activated cell sorter analysis of mESC cultured in GS and 2 h in DM prior (control vector LCv2_NTC) and after CRISPR/Cas9 inactivation of eNOS (LCv2_eNOS_1 or LCv2_eNOS_2). Left cytograms: representative scatter plots showing the forward (FSC-A) and side scatter (SSC-A) distribution of the mESC population. Right panels: representative dot plot showing the fluorescein isothiocyanate (FITC) and SSC-A distribution of the mESC population in the presence of DAF fluorescent probe. b qRT-PCR analysis of mRNA expression relative to Wnt3, Dll1, Wnt7b, Mesp2, and Tbx4 transcripts in mESC cultured for 24 h in GS and DM prior (control vector LCv2_NTC; black bar) and after CRISPR/Cas9 inactivation of eNOS (LCv2_eNOS_1/_2; gray bars). Data are shown as the mean of three independent experiments for each CRISPR/Cas9 vector ± s.e.m. represented as fold increase compared to control mESC after subtraction of the housekeeping gene p0 signal (*p < 0.05 DM vs. GS; °p < 0.05 LCv2_eNOS_1/_2 vs. LCv2_NTC). c Representative western blot performed with cell extracts obtained from mESC infected by Ad_GFP, Ad_eNOS^wt, or Ad_eNOS^S1177D cultured for 24 h in GS or DM (n = 3 each group). The expression of eNOS and cMyc was evaluated. The signal from α-tubulin antibody was used as loading control. Full-length blot provided in Supplementary Fig. 10a. d Representative immunoprecipitation (IP) western blotting (WB) analysis of Hdac2 S-nitrosylation performed in mESC infected by Ad_GFP, Ad_eNOS^wt, or Ad_eNOS^S1177D cultured in GS or after 2 h from release into DM (n = 3 each group). Full-length blot provided in Supplementary Fig. 10b. e qRT-PCR analysis of mRNA expression relative to Wnt3, Wnt7b, Tbx4, Dll1 and Mesp2 transcripts in mESC cultured for 24 h in GS prior (control Adeno virus Ad_GFP; black bar) and after infection by Ad_eNOS^wt(gray bars) or Ad_eNOS^S1177D expressing the constitutive active eNOS (white bars). Data shown as the mean of three independent experiments ± s.e.m. represented as fold increase compared to Ad-GFP infected mESC after subtraction of the housekeeping gene p0 signal (*p < 0.05 vs. Ad_GFP). Data analyzed by Kolmogorov–Smirnov test