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. 2020 Jan 21;10:828. doi: 10.1038/s41598-020-57663-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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Astrocytes aged in vitro develop a senescence-like phenotype. (A) Analysis of mRNA expression for the senescence-associated genes p16^INK4A, p21, p53, Il-6, Timp-1, and Mmp-3 by qPCR in young (white) and aged (purple) astrocyte cultures. (B) Expression of senescence-related genes p16^INK4A, p21, and p53 following rapamycin treatment (25 nM/day, 72 h) Fold expression determined by normalization to expression in young astrocytes. Western blot analyses of (C) p21, (D) HMGB1, (E) TGFB1 and the intermediate filament protein (F) GFAP from young and aged astrocyte cell lysates. Densitometry (a.u.) for each factor was used to determine expression relative to β-actin. Representative immunocytochemistry for (G) p16^INK4A and (H) p21 in young and aged astrocytes. Scale bar, 20 μm. (I) Representative SA-β-gal staining of young and aged astrocyte cultures, and (J) quantification of SA-β-gal staining in quadruplicate independent cultures. Scale bar, 20 μm. (I) Significance as indicated where: (A) **P < 0.01, ***P < 0.001, and ****P < 0.0001, Student’s t-test with Welch’s correction. (B) *P < 0.05, **P < 0.01, Student’s t-test with Welch’s correction. (C–F) *P < 0.05, **P < 0.01, and ***P < 0.001, Student’s t-test with Welch’s correction. (J) ****P < 0.0001, Student’s t-test with Welch’s correction. Values are expressed as mean ± SEM.