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. 2020 Dec 7;10:21364. doi: 10.1038/s41598-020-78206-4

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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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ERK is required for fibrous tissue marker regulation but not required to inhibit chondrogenesis. (A) Sclerotome was treated with a MEK inhibitor PD184352, PD, to inhibit ERK activity, for 24 h and then cells were treated with TGFβ1 for 8 h. Immunoblot was used to determine relative levels of pERK1/2, ERK1/2, and α-tubulin. (B) Blots were quantified using ImageJ and Graphpad Prism. Activity of ERK was determined as the ratio of pERK over ERK under the indicated conditions. (*) indicates significance, p < 0.05, n = 3. (C, D) RNA was isolated from cells under the indicated conditions and qPCR was used to determine the relative expression of (C) Scx, Fmod and Adamtsl2 mRNA or (D) Ebf1 and cMAF mRNA. mRNA levels were normalized to Hprt. Expression is shown relative to the untreated control. Data analyzed by REST software. (*) indicates significance, p < 0.05, n = 3. Detailed results from qPCR REST analysis are shown in Tables S9, S10. Immunoblots were cropped for clarity. Examples of uncropped blots are found in Supplementary Figures.