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. 2020 Dec 21;11:6422. doi: 10.1038/s41467-020-20222-z

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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 Brightfield images and MLL1 immunofluorescence (green) of control and 6d doxycycline-induced shMLL1 human Ls174T spheres, scale bar 100 µm, magnification of brightfield images 25 µm, immunofluorescence 20 µm. Quantification of spheres >50 µm in size on the right, n = 3 biologically independent samples, two-tailed unpaired t-test, **p = 0.0013. Data are presented as mean values ± SD. b Xenografts of control and shMLL1 Ls174T cells in NMRI^nu/nu mice. Mice inoculated with shMLL1 cells were administered doxycycline from day −1 throughout the experiment. Left: tumor growth curves plotted as median with range, n = 6 independent animals inoculated with three biologically independent induced or non-induced shMLL1 Ls174T cell clones in duplicates over two independent experiments, two-tailed unpaired t-test, d16 **p = 0.007, d21 ***p = 0.0034, d23 ***p = 0.00016, d28 ***p = 0.00018. Right: dissected tumors at day 28, scale bar 2 cm. c Differentially expressed genes in shMLL1 human Ls174T cells, cut-offs log₂ fold-change ≥ 0.5, adjusted p-value ≤ 0.05, compared to intestinal stem cell signatures reported by the groups of Clevers, Soshnikova, and Batlle. The correlation table indicates the number of common genes, the Jaccard index of similarity is encoded in green color shadings (lighter means more similar). d Volcano plot of differentially expressed genes in shMLL1 human Ls174T cells (blue dots), overlaid with MLL1-regulated colon cancer stem cell signature (Supplementary Data 3), marked as green dots and indicated by name. e mRNA expression of intestinal stem cell genes and classical Wnt targets in control and 6d doxycycline-induced shMLL1 human Ls174T spheres, n = 4 samples derived from three biologically independent cell clones (IGFBP4, OLFM4 n = 3), two-tailed unpaired t-test, LGR5 ***p = 0.001, IGFBP4 **p = 0.0018, SMOC2 *p = 0.015, OLFM4 *p = 0.03. Data are presented as mean values ± SD. f Tcf/Lef luciferase reporter assays in control and shMLL1 cells treated with doxycycline for 10 days, n = 6 samples over three independent experiments, two-tailed unpaired t-test. Wnt-responsive luciferase activity (TOPflash) calculated relative to control (FOPflash). 3 µM CHIR99021 and 50 µM LF3 were added for 24 h to stimulate and repress Wnt signaling, respectively, n = 3 independent experiments, two-tailed unpaired t-test, ***p = 0.0002, **p = 0.0074. Data are presented as mean values ± SD. Source data are provided as a Source Data file.