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. 2020 Oct 20;10:17853. doi: 10.1038/s41598-020-74944-7

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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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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Inhibition of VSMC phenotypic switching with neutralizing IL11 antibodies or ERK inhibition. (a) Proliferation (EdU^+ve staining) and COL1A1 expression after TGFβ1 or ANGII stimulation in the presence of isotype control (IgG) or IL11 (X203) antibodies. Representative immunostaining images and Operetta assay results from 4 independent experiments. (b) Total collagen secretion (Sirius Red) following TGFβ1 or ANGII stimulation with either IgG or X203 antibody (n = 4). (c) Cytokine-induced VSMC migration (scratch wound assay) in the presence of IgG or X203 (n = 4). (d) Matrigel invasion indices of TGFβ1-stimulated VSMC in the presence of X203 or IgG antibody (n = 3). (e) Representative immunoblots of IL11-induced ERK1/2 and STAT3 phosphorylation in human VSMCs. Samples were derived from the same experiment and blots were processed in parallel. Extended blots are presented in Supplementary Fig. S12 online. Collated densitometry for phosphorylated ERK1/2 and STAT3 normalized to total ERK1/2 and STAT3 respectively (n = 9). P-values stated in comparison to 0 min time point. (f) Representative immunoblots of ERK1/2 activation and extracellular matrix proteins fibronectin (FN1) and collagen 3 (COL3A1) in human VSMCs stimulated with TGFβ1, or ANGII in the presence of anti-IL11 (X203) or IgG control antibodies. Samples were derived from the same experiment and blots were processed in parallel. Extended blots are presented in Supplementary Fig. S12 online. Collated densitometry for phosphorylated ERK1/2 normalized to total ERK1/2, FN1 and COL3A1 normalized to GAPDH (n = 5). (g) Proliferation (EdU^+ve cells) and COL1A1 expression after IL11 stimulation and either MEK/ERK inhibitor U0126 (10 µM) or dimethyl sulfoxide (DMSO) vehicle. Representative immunostaining images and Operetta assay results from 4 independent experiments. Scale bars in representative images for a represent 200 µm and g represent 100 µm. Statistical analyses by one-way ANOVA with Sidak post hoc tests or two-tailed paired t-test; data presented as mean ± SD. Cytokines were applied for 24 h at doses of 5 ng/ml for IL11 and TGFβ1 and 100 nM for ANGII and antibodies at 2 µg/ml.