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. 2020 Jul 30;10:12864. doi: 10.1038/s41598-020-69737-x

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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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Class I HDAC isoforms are overexpressed in PAH and are associated with adventitial fibroblast proliferation. Cell proliferation was assessed by BrdU incorporation in (A) donor- and IPAH-PAAFs (n = 3) cultured ex vivo. Data was normalized to respective donors. (B) qPCR analysis of HDACs was performed on the RNA isolated from the PAAFs harvested from healthy donors (n = 4) and PAH (n = 4) patients. ∆C_t values were normalized to β-2 microglobulin (β2M) and further normalized (∆∆C_t) to donor controls. (C) Western blots were performed on lysates extracted from human PAAFs of healthy donors (n = 4) and PAH (n = 4) patients. (D) Blots were quantified by densitometry and are represented as box plots after normalization to internal loading control. (E) HDAC activity (OD/min/mg) was measured in PAAFs harvested from healthy donors (n = 3) and PAH (n = 3) patients using colorimetric assay kit (Epigenase HDAC Activity/Inhibition Assay Kit, Epigentek). (F) Overexpression of HDACs was achieved by transient transfection of donor-PAAFs (n = 3) with validated HDAC1, HDAC2, HDAC8 plasmids, and their functional effects on proliferation was assessed by BrdU incorporation, 48 hours (h) post-transfection. Empty vector plasmid was used as negative control. Data was normalized to untransfected cells. In all plots, significant differences between corresponding comparisons are indicated as *p < 0.05, Student's t-test.