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. 2018 Oct 8;8:14966. doi: 10.1038/s41598-018-33378-y

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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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Validation of positive hits identified from the screening. U2OS cells coexpressing streptavidin-KDEL and ss-SBP-GFP were treated with selected agents at 10, 20 and 40 μM for 2, 4, or 6 h before biotin treatment. Four hours after exposure to biotin, cells were fixed for following image acquisition and analysis (A). Images were segmented and average cytoplasmic GFP intensity was quantified. The data was normalized by Z-scoring and subjected to hierarchical clustering (C). Verified agents were further subjected to an ELISA assay to measure ss-SBP-GFP concentration in supernatants. U2OS coexpressing streptavidin-KDEL and ss-SBP-GFP cells were treated with the selected agents validated as described above at the indicated concentrations for 4 h before addition of biotin, and cell culture supernatants were collected after 1 or 4 h of incubation for ELISA (B). The quantity of ss-SBP-GFP in supernatants was normalized to cellular lysates. Cells incubated in the absence of biotin were used as negative controls. Data is reported as mean ± SEM (n = 3) (D). Statistical analysis was performed by means of t test, ***p < 0.001 as compared to cells treated with biotin alone.