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. 2017 Oct 10;7:12916. doi: 10.1038/s41598-017-13365-5

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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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Aconitase inhibition does not protect palmitate-induced lipotoxicity. INS-1 cells were treated with different concentrations of sodium fluorocitarte (SFC) or trans-aconitic acid (TAA) for 8 h. (a,b) Aconitase activity was measured by aconitase activity assay kit. (c) Cell viability was measured by MTT assay. Data shown are relative activities. Data are expressed as mean ± SE and analyzed by one-way ANOVA. *p < 0.05; **p < 0.01; ***p < 0.001 vs. PBS-treated INS-1 cells. ^$$ p < 0.01 vs. 0.2 mM SFC-treated INS-1 cells. (d) INS-1 cells were treated with 0.4 mM palmitate (PA) for 16 h in the presence of different concentrations of TAA. Cleaved caspase 3 levels were investigated by immunoblotting with anti-caspase 3 antibodies. Data are presented as mean ± SE and analyzed by one-way ANOVA. (e,f,g) INS-1 cells were transfected with siRNA of green fluorescent protein (GFP), aconitase 1 (Aco1), aconitase 2 (Aco2), or aconitase 1/aconitase 2 (Aco1, 2). After 36 h of incubation, knockdowns of aconitases were determined by immunoblotting with anti-Aco1 or Aco2 antibodies. Aconitase activities were measured by aconitase activity assay kit. Transfected INS-1 cells were treated with 0.4 mM palmitate for indicated time period. Cell death was determined by measuring levels of cleaved caspase 3 in immunoblotting with anti-caspase 3 antibodies. Full-size original blots are presented in supplementary Fig. S6. Data are presented as mean ± SE and analyzed by unpaired Student’s t-test (Immunoblotting for aconitases and activity for aconitases) or two-way ANOVA (Immunoblotting for cleaved caspase 3). *p < 0.05; **p < 0.01; ***p < 0.001 vs. siGFP-transfected INS-1 cells.