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. 2020 Jul 8;10:11207. doi: 10.1038/s41598-020-67466-9

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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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Mutation of residues in DimDAF-12 ligand binding pocket abolishes the activity of the receptor. NIH3T3 cells were co-transfected with wild type Gal4-DimDAF-12_LBD or the corresponding mutants and luciferase gene reporter construct and then tested with 0.01 or 0.1 uM of Δ4- (A single mutant, B double mutants) or Δ7- (C single mutant, D double mutants) dafachronic acids (DAs). The ligand binding and transactivation activity were assessed by measuring the luciferase activity, normalized by Renilla luciferase activity for transfection efficiency and expressed as relative light units (RLU). Data represent the average of normalized luciferase activity and the error bars correspond to the standard error of the mean from a triplicate assay. The figure shows a representative experiment performed in triplicate. The significance of the effects of mutation on DimDAF-12 LBD activity was analyzed by two-way ANOVA with multiple comparisons without correction using Prism 6.0 (Graph Pad Software, Inc.) (***p < 0.0005; **p < 0.005; *p < 0.05) versus wild-type.