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. 2020 Apr 27;10:7058. doi: 10.1038/s41598-020-63996-4

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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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DHR treatment shifts CAFs into quiescent fibroblasts. (a,b) Flow cytometry analysis of apoptotic cell death in SCAF#36 cells after two weeks of 0.1 μM or 1 μM DHR treatment in medium containing 1% serum. (a) Treated cells were stained with annexin V-APC/propidium iodide, and apoptotic cell death was analyzed by flow cytometry. (b) Cells that underwent early apoptotic cell death (lower right quadrant) and late apoptotic cell death (upper right quadrant) were calculated as a percentage of all cells. (c–e) SCAF#36 cell proliferation, viability, and cytotoxicity under three days of 0.1 μM or 1 μM DHR treatment. (c) SCAF#36 cells were treated with 0.1 μM or 1 μM DHR, and cell proliferation was counted daily. Cell growth was normalized to day 0. Symbols represent the means ± SEM of five independent experiments. Differences were evaluated by two-tailed student’s t-test. **P < 0.01. (d) After 72 hours of DHR treatment, cell viability was assessed using an EZ-cytox kit. The optical absorbance measurement of formazan at 450 nm was assumed to be proportional to the number of viable cells in each well. Symbols represent the means ± SEM of three independent experiments. (e) The supernatants from SCAF#36 cells treated with DHR for 72 hours were assessed for LDH release with an EZ-LDH cytotoxicity assay kit. Experiments were done in triplicate. Bars represent the means ± SEM of three independent experiments. Differences were evaluated by two-tailed student’s t-test. (f) The effects of DHR treatment on the colonization of CAFs. SCAF#36 cells were plated, and the number of colonies was counted. Cells were either treated with 0.1 μM or 1 μM DHR every three days during colonization for two weeks (left panels) or pretreated for three days before colonization (right panels). Representative images are shown. Experiments were done in triplicate. Bars represent the means ± SEM of three independent experiments (treatment) or two independent experiments (pretreatment). Differences were evaluated by two-tailed student’s t-test. **P < 0.01.