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. 2017 Jun 14;7:3473. doi: 10.1038/s41598-017-03152-7

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© The Author(s) 2017

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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HDAC3 modulates expression of AQP5. NIH3T3 cells were co-transfected with 358-Aqp5-Luc, Sp1 and increasing amounts of HDAC1 (A), HDAC2 (B) and HDAC3 (C) expression vectors or empty vector (pCDNA3). HDAC2 and HDAC3, but not HDAC1 inhibited Sp1-activated Aqp5 transcription. Protein concentrations were used for sample normalization (n = 3, *p < 0.05 compared to empty vector). WB (D) and corresponding quantitative analysis (E–G) show that AQP5 expression (normalized to GAPDH (37 kD)) is significantly upregulated (D,G) following knockdown of HDAC3 (59 kD) with shRNA (D,E) but not HDAC2 (49 kD) with siRNA (D,F) (n = 3, *p < 0.05 compared to non-silencing shRNA and GFP siRNA) in MLE-15 cells. (H) ChIP with anti-acetyl-H3 Ab (H3Ac) demonstrates enrichment of H3 acetylation at the Aqp5 promoter/enhancer region homologous to the proximal 358-bp of the rat Aqp5 promoter following HDAC3 knockdown in MLE-15 cells (n = 3, *p < 0.05). ChIP efficiency was calculated relative to non-silencing shRNA (NS-shRNA)-treated cells precipitated with H3Ac Ab, which was set as 1. Rabbit IgG pull-down is used as control.