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. 2019 Jul 23;9:10681. doi: 10.1038/s41598-019-47128-1

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© The Author(s) 2019

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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PAM induces DNA damage and apoptosis mainly in OS cells. We used different treatment times (5, 10 and 15 min) in PAM + Pyr at 1 L/min and 10 mm gap to analyze the cytoprotective effect of Pyr. (A) Analysis of apoptosis/necrosis by FACS using AnnexinV/PI on hBM-MSCs (top) and SaOS-2 (bottom). The percentage of apoptotic SaOS-2 on PAM + Pyr increases from the control (5.4%): 5 min (6.7%), 10 min (10.5%) and 15 min (19.2%). Representative data at 24 hours are shown. Analysis were done in duplicates. (B) Representative images (left) and quantification (right) of immunofluorescence for γH2AX (Green) and DAPI (blue) after 2 h on (B) SaOS-2 and (C) hBM-MSCs treated with PAM +/− Pyr at 1 L/min and 10 mm for 10 min. Quantitative analysis are shown as percentage of γH2AX positive (green) vs the total number of DAPI (blue) positive nuclei at the indicated treatment times. PAM +/− Pyr on SaOS-2 induces no statistically significant differences exist at 10 (p value = 0.1573) and 15 min (p value = 0.008), showing only significant differences on 5 min treatment time (p value = 0.0008). PAM -Pyr is capable to affect DNA integrity on healthy cells (p value = 0.0002). Scale bar = 50 µM. Data are presented as mean, n = 3. Error bars represent the SD, and asterisks indicate statistically significant differences between the PAM +/− Pyr series (*p < 0.05; **p < 0.01, ***p < 0.001; two-sided Student’s t-test).