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. 2019 Jun 17;9:8671. doi: 10.1038/s41598-019-45191-2

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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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Acquisition of a senescent phenotype by CAP He treated ASC and dermal fibroblatsts. (A) ASC and dermal fibroblasts were treated (CAP or NT) and nuclear p16 was imaged by immunofluorescence. Images were acquired using the high content imaging system Operetta (Perkin Elmer, lens x20) to quantify p16 nuclear spots number per cell and fluorescence intensity per cell (means of 5 fields). (B) Senescence associated-morphological change was observed 7 and 14 days after treatment. Cells morphology was evaluated by DAPI (nuclear) and β-tubulin (cytoskeleton) labeling using the high content imaging system Operetta (Perkin Elmer, lens x20). Pictures were taken with a Nikon, Eclipse, TE2000-S microscope (x200 magnification) (n = 4). (C) Nuclear size was additionally quantified (n = 4). (D) Senescence associated-β-galactosidase (SA-β-gal) was quantified 5 and 7 days after treatment by flow cytometry with a substrate (C12-FDG). Quantification is represented by the Mean of Fluorescence Intensity (MFI) normalized to the same condition without labelling. SA-β-gal was also evaluated by biochemical detection (blue staining) and pictures were taken at day 7 with a Nikon, Eclipse, TE2000-S microscope (ASC, n = 3 to 4; fibroblast, n = 3 to 5). (E) Senescence associated cytokine secretion was evaluated in treated cells supernatant for IL-6 and IL-8 by ELISA (n = 3 to 6). Data represent mean ± SEM of independent experiments as indicated (n).