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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. 3 — Endogenous NO synthesis and Zeb1-Hdac2 complex formation. a cGMP quantification in GS (black bar), in DM ± PTIO, ODQ, L-NAME (gray bars) for 1, 2, or 3 h and in NO ± PTIO (white bars) for 24 h. (n = 3; *p < 0.05 DM vs. GS; °p < 0.05 treatments vs. DM). b Zeb1-chromatin binding analysis on: Wnt3, Wnt7b, Tbx4, Dll1, and Mesp2. Chromatins extracted in DM (gray bars) or DM + PTIO (striped bars). Data compared to IgG value (striped bars) after Input normalization (n = 3). c Left panel: representative immunoprecipitation (IP)/western blotting (WB) analysis of Hdac2 S-nitrosylation in GS (24 h), DM or NO (2 h). Right panel: densitometry (n = 3; *p < 0.05 NO vs. DM). Full-length blot provided in Supplementary Fig. 9a. d Reciprocal co-IP analysis showing Zeb1:Hdac2 (left) and Hdac2:Zeb1 (right) complex formation in GS, DM or NO (24 h; n = 3 each group). Full-length blot provided in Supplementary Fig. 9b and c. e Left panel: representative co-IP analysis showing Zeb1:Hdac2^wt and Zeb1:Hdac2^C262A/C274A complex formation compared to empty vector (Ev)-transfected HeLa cells. HeLa transfected with Ev or co-transfected with pCMV6_Zeb1/pCIG2_Hdac2^wt or pCMV6_Zeb1/pCIG2_Hdac2^C262A/C274A. Right panel: densitometry (n = 3; *p < 0.05 Zeb1:Hdac2^wt vs. Ev; °p < 0.05 Zeb1:Hdac2^C262A/C274A vs. Zeb1:Hdac2^wt). Full-length blot provided in Supplementary Fig. 9d. f Left panel: representative WB analysis of eNOS, nNOS, and iNOS in GS or DM (n = 3). Loading control: α-tubulin. Full-length blot provided in Supplementary Fig. 9e. Right panel: eNOS, nNOS, and iNOS mRNA analysis in GS (black bars) or DM (gray bars). Data expressed as fold increase of GS after subtraction of the housekeeping gene p0 signal (n = 3; *p < 0.05 DM vs. GS). g Zeb1-chromatin binding analysis on eNOS promoter in GS (black), DM (gray), and NO (white). Data represented as fold increase of GS after input normalization (n = 3). h, i eNOS mRNA expression analysis in GS, DM, and NO prior (control vector LCv2_NTC; black bar) and after CRISPR/Cas9 inactivation of Zeb1 (h; LCv2_Zeb1_1/_2; gray bars) or CRISPR/Cas9 inactivation of Hdac2 (i; LCv2_Hdac2_1/_2; white bar). Data represented as fold increase compared to control after subtraction of the housekeeping gene p0 signal (n = 3; *p < 0.05 LCv2_Zeb1_1/_2 or LCv2_Hdac2_1/_2 vs. LCv2_NTC). Data represented as mean ± s.e.m. and analyzed by Kolmogorov–Smirnov test