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. 2020 Aug 18;43(6):1161–1174. doi: 10.1007/s13402-020-00549-x

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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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — Macrophages induce EMT of pancreatic cancer cells in a PAR1-dependent manner. (A) Phase-contrast microscopic images of PANC-1 wildtype cells treated with control or M0-CM medium. PAR1 was inhibited by Vorapaxar (500 nM) using DMSO as a mock control. Spindle-shaped cells were quantified using the cell counter tool of Image J, after which the percentage per field was calculated according to the total number of cells. Shown is the mean ± SEM (n = 3); Student’s t test. (B) Morphology assessment of shCtrl and shPAR1 PANC-1 cells treated with M0-CM. Spindle-shaped cells were quantified using the cell counter tool of Image J, after which the percentage per field was calculated according to the total number of cells. Shown is the mean ± SEM (n = 3); Student’s t test. In panels A and B, magnification is 20x, and the scale bar indicates 50 μm. (C-D) Relative mRNA expression of CDH1, ZEB1 and VIM in RPMI-1640 (white) or M0-CM (blue) treated PANC-1 (C) and MIA PaCa-2 (D) cells. PAR1 was inhibited by Vorapaxar (500 nM) using DMSO as a mock control. Shown is the mean ± SEM (n = 4); Student’s t test. (E-F) Relative mRNA expression of CDH1, ZEB1 and VIM in RPMI-1640 (−) or M0-CM (+) treated PANC-1 (E) or MIA PaCa-2 (F) shCtrl (white) and shPAR1 (gray) cells. Shown is the mean ± SEM (n = 4); Student’s t test. Relative expression levels, as depicted in panels C-F, were calculated using the comparative threshold cycle (dCt method) and normalized to the expression of the reference gene TBP