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. 2017 Jun 27;7:4292. doi: 10.1038/s41598-017-04612-w

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© The Author(s) 2017

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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ACF Binds BSG and Inhibits the MCT/BSG Interaction. (A) Split Renilla luciferase (Rluc) system for identifying inhibitors of MCT/BSG Interaction. N-terminal and C-terminal portions of Rluc were attached to MCT (MCT1 and MCT4) and BSG, respectively. (B) The ratio of Renilla/Firefly luciferase activity (Rluc/Fluc) was determined using HEK293 cells co-transfected with pCDNA3, which encodes Fluc, with the indicated constructs. Each value was then normalized to the results for an empty vector. Data represents mean ± SD (n = 3). **P < 0.01 (Student’s t-test) vs. samples lacking BSG. (C) Dose dependency curve for NhRL-MCT4 and BSG-ChRL interaction. IC50 = 4.6 μM. (D,E) Surface plasmon resonance (SPR) analyses were used to determine the equilibrium dissociation constant (KD) between Basigin and ACF. Sensorgrams (D) and a fitting curve (E) are shown. Experiments were performed in triplicates and comparison was made between native BSG and 6X-HIS BSG with similar KD values of 0.16+/−0.07 and 0.23 +/− 0.1, respectively. (F) A representative cellular thermal shift assay (CETSA) analysis for U87-MG GBM cells treated with ACF or vehicle (DMSO) showing ACF increases BSG’s melting temperature, therefore, providing further evidence for direct drug/target interaction in live cells. (G) A representative western blot analysis of CETSA assay.