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. 2020 Jan 15;10:357. doi: 10.1038/s41598-019-57047-w

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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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SOX9 silencing impairs tumor cell survival, induces senescence and abrogates proliferation in cancer cells. (A) Representative Western blots of SOX9 protein expression in MKN45 and AGS GC cell lines, Panc-1 and RWP-1 PDAC cell lines, and U373 and U251 GBM cell lines lentivirally transduced with a specific shRNA against SOX9 (sh1) or the corresponding control vector (pLKO) (n ≥ 4). β-actin levels were used as a loading control. (B) Cell growth at day 5 (percentage of cells) in SOX9-silenced (sh1) cells respect to control (pLKO) cells (n ≥ 4). (C) Apoptosis represented by the percentage of active Caspase-3 positive cells in SOX9-silenced (sh1) cells and the corresponding control cells (pLKO) determined by immunofluorescence staining (n ≥ 4). (D) Representative image of active Caspase-3 positive cells in RWP-1 control (pLKO) and SOX9-silenced (sh1) cells. (E) Apoptosis represented by the percentage of cleaved PARP1 positive cells in SOX9-silenced (sh1) cells and the corresponding control cells (pLKO) determined by immunofluorescence staining (n ≥ 4). (F) Representative image of cleaved PARP1 in RWP-1 control (pLKO) and SOX9-silenced (sh1) cells. (G) Senescence represented as the quantification of the percentage of β-Galactosidase (SA β-Gal) positive cells in SOX9-silenced (sh1) cells and the corresponding control cells (pLKO) (n ≥ 4). (H) Representative images of SA β-Gal staining in RWP-1 (left) and U251 (right) control (pLKO) and SOX9-silenced (sh1) cells. (I) Proliferative capacity represented by the percentage of phospho-histone H3 (p-H3) positive cells (n ≥ 4) determined by immunofluorescence staining in control (pLKO) and SOX9-silenced (sh1) cells. (J) Representative image of p-H3 in RWP-1 control (pLKO) and SOX9-silenced (sh1) cells. Asterisks (*, ** and ***) indicate statistical significance (p < 0.05, p < 0.01, and p < 0.001, respectively).