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. 2017 Aug 3;7:7207. doi: 10.1038/s41598-017-07455-7

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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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The environmental effect of liquid culture is upstream of known genetic components and induces similar pathways. (A) Mouth-form ratios of eud-1 overexpression²⁷ and a Ppa-nhr-40 ²⁸ mutant in liquid culture reveals no effects, suggesting these genes are downstream, n = 3 biological replicates. (B) Addition of 1 µM dasc#1 exhibits a variable response that appears to induce Eu, although it is not statistically significant (p = 0.068). (C) Expression analysis of four genes by RNA-seq from eud-1 mutants (the average of 4 homozygous mutant alleles is represented)²⁷ (100% St) compared to the RS2333 California strain (70–100% Eu), y-axis = fpkm (relative expression). (D) Reverse transcription-quantitative PCR (RT-qPCR) of P. pacificus PS312 grown in liquid culture/S-medium (LC) vs. NGM-agar plates (AG) for the two biological replicates displayed, with four technical replicates each. The y-axis represents 2^ΔCt (relative expression) compared to the housekeeping gene Ppa-Y45F10D.4 (iron binding protein)⁶⁹, error bars represent standard deviation of n = 4 technical replicates.