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. 2018 Sep 4;8:13180. doi: 10.1038/s41598-018-31570-8

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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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(A) Wavelength dependent ROS production. DCF-DA assay of PAC-2 cells exposed during 4 hours to different wavelengths of light. Blue, red and grey bars indicate ROS levels assayed upon blue, red and white light exposure, respectively. The means of fold induction (n = 24) ± SD with respect to time 0 are plotted on the y-axis, while the duration of light exposure is plotted on the x-axis. Levels of significance between peak points of DCF-DA fluorescence and time 0 are calculated by t-test and are indicated (***p < 0.001, **p < 0.01, *p < 0.05). (B–D) ROS-dependent, light-induced clock gene expression in PAC-2 cells. qRT-PCR analysis of expression of the light inducible genes zfper2 (dark blue bars) and zfcry1a (light blue bars) in PAC-2 cells during blue light exposure in the presence of different concentrations of (B) NAC, (C) DPI and (D) VAS 2870. Samples were harvested after 3 hours of light exposure. On the y-axis is plotted the mean ± SD of mRNA fold induction with respect to the control samples incubated with the corresponding inhibitor-vehicle. The specific concentrations of NAC, DPI and VAS 2870 used are indicated on the x-axis of each graph. Levels of significance obtained in three independent experiments between the vehicle (0) and each inhibitor concentration are calculated by t-test and indicated (***p < 0.001, **p < 0.01, *p < 0.05).