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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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BMI1 re-expression in cancer cells with SOX9 silencing restores the aggressive phenotype conferred by SOX9 in vitro. (A) Representative Western blots of SOX9, BMI1, p21^CIP and GFP protein expression in MKN45, Panc-1 and RWP-1 control (pLKO) and SOX9-silenced cells (sh1) lentivirally transduced with BMI1 (pLKO BMI1 and sh1 BMI1) or GFP (pLKO GFP and sh1 GFP)(n ≥ 4). β-actin levels were used as a loading control. (B) Relative cell growth determined by cell count experiments in RWP-1 pLKO and SOX9-silenced PDAC cell line (sh1) lentivirally transduced with BMI1 (pLKO BMI1 and sh1 BMI1) or GFP (pLKO GFP and sh1 GFP) (n ≥ 3). (C) Apoptosis represented by the percentage of active Caspase-3 positive cells determined by immunofluorescence staining in MKN45, Panc-1 and RWP-1 pLKO and SOX9-silenced cells (sh1) lentivirally transduced with BMI1 (pLKO BMI1 and sh1 BMI1) or GFP (pLKO GFP and sh1 GFP) (n ≥ 4). (D) Apoptosis represented by the percentage of cleaved PARP1 positive cells determined by immunofluorescence staining in MKN45, Panc-1 and RWP-1 pLKO and SOX9-silenced cells (sh1) lentivirally transduced with BMI1 (pLKO BMI1 and sh1 BMI1) or GFP (pLKO GFP and sh1 GFP) (n ≥ 4). (E) Cellular senescence represented by the percentage of β-Galactosidase (SA β-Gal) positive cells in Panc-1 and RWP-1 pLKO and SOX9-silenced cells (sh1) lentivirally transduced with BMI1 (pLKO BMI1 and sh1 BMI1) or GFP (pLKO GFP and sh1 GFP) (n ≥ 4). (F) Proliferative capacity represented by the percentage of phospho-histone H3 (p-H3) positive cells analyzed by immunosfluorescence staining in MKN45, Panc-1 and RWP-1 pLKO and SOX9-silenced cells (sh1) lentivirally transduced with BMI1 (pLKO BMI1 and sh1 BMI1) or GFP (pLKO GFP and sh1 GFP) (n ≥ 4). Asterisks (*, ** and ***) indicate statistical significance (p < 0.05, p < 0.01, and p < 0.001, respectively).