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. 2018 Apr 10;8:5723. doi: 10.1038/s41598-018-24152-1

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© The Author(s) 2018

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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S. pneumoniae-dependent KLF4 expression is induced by viable, replicating bacteria which undergo autolysis and are in direct contact to the host cells. Wt BMMs were stimulated with 10⁶ CFU/ml viable (R6x and NaIO₄-inactivated R6x) or 10⁷ CFU/ml dead (heat- (hi) and ethanol-inactivated (eth) R6x) pneumococci (a). The need of bacterial replication was tested by a stimulation of wt BMMs with 10⁶ CFU/ml R6x in RPMI with or without 10% FCS (c). Wt BMMs were stimulated with 10⁶ CFU/ml R6x in direct contact or separated using a transwell system (e) or with R6x and R6xΔlytA (10⁶ CFU/ml each) (g). Cell lysates were collected 6 h after stimulation and analysed for KLF4 expression. Actin was used to confirm equal protein load. pp38 proved stimulation efficiency (e). All blots are representatives out of 3 independent experiments with similar results. The densitometry of the KLF4 and actin bands of the blots was quantified using Odyssey 2.0 infrared imaging system. The ratio between the KLF4 and actin densitometry was calculated. (b) shows the quantification of the blots of experiment (a), (d) for (c), (f) for (e) and (h) for (g). Data represents mean of 3 independent experiments. Differences were indicated as follows: **p < 0.01; ***p < 0.001.