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. 2019 Nov 1;10:5011. doi: 10.1038/s41467-019-13028-1

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© The Author(s) 2019

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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Fig. 4 — FASN deletion leads to ATP citrate lyase (ACLY) inhibition, blocking 3D growth. a Representative pictures of the spheroids recovered from the different genotypes growing under ultralow attachment conditions for 72 h (left). Spheroid diameter (n = 30 spheroids, FASN^lox/lox-PyMT; n = 24 FASN^∆/∆-PyMT) and number of cells over 72 h (right). Scale bars, 500 μM. Presented data are the mean values ± SD. ***P < 0.001; Student’s t test. b OCR assay performed with cells recovered from the previous experiment and monolayer cultures. Oligomycin, FCCP, and antimycin/rotenone were added at the indicated time point. Data are represented as the means ± SEM. c Representative flow cytometry analysis showing constitutive mitochondrial superoxide levels in monolayer compared with 3D conditions in PyMT MEFs loaded with MitoSOX Red (FASN^lox/lox-PyMT, n = 4; FASN^∆/∆-PyMT, n = 5). The quantification of MitoSOX Red fluorescence intensity is shown on the left. Presented data are the mean values ± SD. ***P < 0.001, Student’s t test. d Quantification of MitoSOX Red fluorescence intensity by flow cytometry analysis in FASN^lox/lox-PyMT, FASN^∆/∆-PyMT, and DMNQ-treated (10 μM; 20 min) FASN^lox/lox-PyMT MEFs (n = 3). e Effect of 10 μM DMNQ treatment in the ability of FASN^lox/lox-PyMT MEFs to form spheroids. Scale bars, 500 μM. Representative pictures for each condition are shown. f Representative pictures showing the effects of sodium citrate (5 mM) or SB204990 (30 μM) in the formation of tumor spheroids by FASN^lox/lox-PyMT MEFs. Scale bars, 500 μM. The lower charts show an increase in ROS production by sodium citrate and SB204990. Representative flow cytometry analysis was shown (n = 4). g Effects in OCR suppression caused by sodium citrate or SB204990 treatment in FASN^lox/lox-PyMT MEFs. Presented data are the mean values ± SEM. Three independent experiments (h) ACLY activity in FASN^lox/lox-PyMT versus FASN^∆/∆-PyMT MEFs. i, j Cell viability was determined in FASN^lox/lox-PyMT MEFs at 24, 48, and 72 h in the presence of various concentrations of SB204990 (n = 8) (i) or sodium citrate (n = 7) (j) under 2D culture conditions. In h–j represented data are the mean values ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001; Student’s t test