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. 2019 Apr 11;9:5912. doi: 10.1038/s41598-019-42040-0

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© The Author(s) 2019

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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TGFβ pathway induction of senescence in the otocyst. (A–D) SAβG staining in isolated otic vesicles from stage HH18 embryos cultured ex vivo for 20 h in free serum medium (0S), in the presence of TGFβ2 (10 ng/ml), SB (SB431542; 10 µM) or a combination of both. (E) Levels of pSMAD2 were measured by western blotting of explanted otic vesicle lysates (two independent experiments). (F) SAβG staining quantification is shown as mean SAβG area ± SEM with respect to the total area, normalized to the 0S condition (white bar). ***p < 0.001 vs 0S; ^#p < 0.05 vs SB. (G) Btg1 and Btg2 mRNA expression levels were measured by RT-qPCR in cultured otic vesicles. Statistical significance was determined with the Student’s t-test: ***P < 0.005, versus 0S. (H) Wnt2b mRNA expression levels were measured by RT-qPCR in non-cultured HH18 and HH19 otic vesicles and in dissected dorsal and ventral regions from HH19 otic vesicles. Statistical significance was determined with the Student’s t-test: ***P < 0.005, versus HH18; ^###P < 0.005, versus HH19 dorsal regions. (I,J) Light microscopy images from TGFβ2-treated otic vesicles showing the relative increment of the endolymphatic duct area (J, asterisk, quantification in K). Quantification for areas were measured and are shown as the mean ± SEM and the statistical significance was determined by Student’s t-test: *P < 0.05, versus control condition (0S, white bars). (L) Wnt2b mRNA levels were measured by RT-qPCR in cultured otic vesicles in 0S medium, in presence of TGFβ2 (10 ng/ml) or palbociclib (1 µM). Statistical significance was determined with the Student’s t-test: ***P < 0.005, versus 0S. Gene expression was calculated as 2^−ΔΔCt and normalized to the levels of 0S (white bars). The results are expressed as the mean ± SEM (three independent experiments performed in triplicate). Ed, endolymphatic duct. Orientation: A, anterior; D, dorsal. Scale bar: 150 μm.