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. 2021 Nov 29;12(12):1118. doi: 10.1038/s41419-021-04407-y

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© The Author(s) 2021

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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Fig. 1 — A Cell proliferation assay of pG-2 cells treated for 48 h with increasing concentrations of a combinatory CAF chemotherapy. The concentrations represent a fraction of the dose used in our previous publication [31]. Cell confluency was assessed every day with a Celigo^®. B Crystal violet staining of pG-2 cells performed on days 2 and 7 after different doses of CAF-chemotherapy. A quantification (absorbance at 590 nm) of solubilized crystal violet at day 7 is provided in the lower panel. C Phase contrast images of pG-2 cells 48 h after CAF-treatment showing a spindle-like morphology characteristic for cells that underwent EMT (scale bar = 250 µm). D Volcano plot showing transcriptome-wide gene expression changes in pG-2 cells treated with CAF for 48 h compared to vehicle. The number of regulated genes regulated upon CAF-treatment in the mRNA-seq analyses treatment was assessed using the following parameters: |log2FC | > 1 and p-adj < 0.05. E Representative GSEA enrichment plots showing significant enrichment of gene signatures characteristic for EMT-processes, stemness traits, and cancer aggressiveness in CAF-treated versus control pG-2 cells. F Heatmap showing the regulation of selected EMT markers identified in the mRNA-seq analyses in CAF-treated versus control pG-2 cells. G and H qRT-PCR results validating the downregulation of epithelial marker (G) and the upregulation of mesenchymal marker (H) upon CAF-treatment of pG-2 cells. Data were calibrated on the control condition and normalized on the Rplp0 housekeeping gene. I: Western blot validating an increase of Vimentin and N-cadherin protein levels as well as a decrease of E-Cadherin protein levels upon 48 h CAF treatment of pG-2 cells. Error bars: standard error of the mean (SEM), *p-val ≤ 0.05, **p-val ≤ 0.01, ***p-val ≤ 0.005, One-way ANOVA: A (AUC: area under the curve), B Student’s t-test: G, H All experiments were performed in at least biological triplicates (n = 3).