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. 2020 Sep 10;11(9):736. doi: 10.1038/s41419-020-02936-6

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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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Fig. 4 — a Tumor organoids derived from Apc/Kras double mutant mice were embedded with adipocytes in 3D Matrigel or treated with BSA, OA, PA, and LA (100 μM each) for 2 days. The levels of Cpt1a expression were analyzed using Western blotting. The relative levels of CPT1A were quantified by normalizing to β-actin and compared to control or BSA-treated cells. b Tumor organoids treated as described in a were analyzed for the expression of Cpt1a mRNA using RT-PCR. Data represents mean ± SD (n = 3, *p < 0.01, ^§p < 0.001, and ^¶p < 0.0001). c The expression of Cpt1a was silenced in Apc/Kras mutant tumor organoids using lentiviral shRNA. Single cell suspensions of control and Cpt1a knockdown cells were seeded in 3D Matrigel. Representative images of control and Cpt1a knockdown tumor organoids are shown after 6 days in culture. Scale bar, 100 μm. d The number of tumor organoids formed and the percentage of organoids showed branching phenotype were quantified (total 1000 cells were seeded per group). Data represent the mean ± SD (n = 3, ^#p < 0.05). e Tumor organoids treated with BSA, OA, PA, and LA (100 μM each) were analyzed for the expression of Wnt β-catenin target genes (including Lgr5, Axin2, and Tcf7) and genes associated with intestinal epithelial cell differentiation (including Krt20 and Muc2) using RT-PCR. Data represents mean ± SD (n = 3, ^#p < 0.05, *p < 0.01, ^§p < 0.001, and ^¶p < 0.0001). f Control and Cpt1a knockdown organoids were subseeded and grown in 3D Matrigel for 3 days. The expression of Cpt1a, Wnt/β-catenin target genes (including Lgr5, Axin2, and Myc), and genes associated with intestinal epithelial cell differentiation (including Krt20 and Muc2) was determined using RT-PCR. Data represent the mean ± SD (n = 3, *p < 0.01 and ^§p < 0.001). g Control and Cpt1a knockdown organoids were treated with OA for 2 days. The mRNA expression of Cpt1a as well as target genes of Wnt/β-catenin (including Lgr5 and Myc) was determined using RT-PCR. Data represent the mean ± SD (n = 3, *p < 0.01).