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. 2018 Nov 30;8:17492. doi: 10.1038/s41598-018-35681-0

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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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(a) In vitro RT-qPCR analysis of target genes following ATRA treatment of ATDC5 cells. Relative genes expression of Eftud2, Gnai3, P4hb, Nedd4 and Parp1 following 12 h and 24 h of 1 µM ATRA treatment. Black bars represented control relative mRNA expression although gray bars shown mRNA expression following 1 µM ATRA treatment. (b) Peptidoglycans coloration of ATD5 cells by Alcian Blue 7 and 21 days after ATRA treatment and in vitro RT-qPCR analysis of targets genes during differentiation. The peptidoglycans accumulation in extra-cellular matrix was used as an index of ATDC5 differentiation into chondrocytes lineage shown by a blue coloration becoming more intense following days. Qualitative analyze suggests an inhibition of ATDC5 differentiation by ATRA. Relative genes expression of Gnai3 and Nedd4 during ATDC5 differentiation, at 7 and 21 days in control condition (no ATRA-treatment). Light turquoise bars represented relative mRNA expression at Day 7 although dark turquoise bars shown mRNA expression at Day 21. (c) In vitro analysis of target genes expression during ATDC5 differentiation and ATRA chronic treatment. Relative genes expression of Eftud2, Gnai3, P4hb, Nedd4 and Parp1 at Day 7 and Day 21 of ATDC5 differentiation. Impact of 1 µM of ATRA chronic treatment on their expression is also analyzed. Black bars represented control relative mRNA expression although gray bars shown mRNA expression following 1 µM ATRA treatment. Quantitative expression of studied genes was determined using the 2−ΔΔCt method. Statistical significance was evaluated using t-test (*P < 0.05; **P < 0.01; ***P < 0.001; n = 4).