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. 2022 Jul 8;13:3971. doi: 10.1038/s41467-022-31663-z

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© The Author(s) 2022

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. 1 — A Gene Set Enrichment Analysis (GSEA) of enriched KEGG pathways in APC-mutant (N = 142), APC/KRAS-mutant CRC (N = 134) as compared to wildtype counterparts (N = 50) from the Cancer Genome Atlas (TCGA) colon and rectal cancer (COADREAD) cohort. Permutation-based p-value. B Terpenoid Backbone Biosynthesis pathway and Steroid Biosynthesis pathway are enriched in APC/KRAS-mutant CRC cases from TCGA cohort. Permutation based p-value. C Fluorescence microscopy of cellular uptake of fluorescent labelled LDL (BODIPY-LDL) in 1CT isogenic cell lines (1CT-K: 1CT-KRAS^G12V, 1CT-A: 1CT-shAPC, 1CT-AK: 1CT-shAPC-KRAS^G12V). Reduced LDL uptake was observed in 1CT-AK cells (n = 6). D Flow cytometry of intracellular fluorescence after incubation with BODIPY-LDL. E Western blot confirmed the up-regulated protein expression of PCSK9 and cholesterol biosynthesis genes in 1CT-AK cells; LDLR was inhibited. F D₂O stable isotope labeling (48 h) of cholesterol and LC-MS analysis of intracellular cholesterol in 1CT isogenic cells, revealing increased de novo cholesterol biosynthesis in 1CT-A and 1CT-AK cells, while having no effect of total cholesterol level (n = 3). G Cell viability of 1CT-AK cells were not sensitive to lipoprotein depletion in culture medium. Cell growth curve of 1CT and 1CT-AK cells in complete or lipoprotein-depleted medium (n = 3). H LC-MS of cholesterol pathway intermediates in 1CT isogenic cells. I LC-MS of cholesterol pathway intermediates in CRC and adjacent normal tissues from 7-weeks-old Apc^min/+Kras^G12D/+Villin-Cre mice (n = 4). J PCR array (Human lipoprotein signaling & cholesterol metabolism; and Human fatty acid metabolism) revealed that KRAS^G12V plus shAPC increased mRNA expression of cholesterol biosynthesis genes. K qPCR validated the increased mRNA expression of PCSK9 and cholesterol biosynthesis genes in 1CT-AK cells compared to 1CT, 1CT-K, and 1CT-A cells, whilst knockout of mutant KRAS in DLD1 cells (i.e., DKS8) exerted an opposite effect (n = 3). L PCSK9 and cholesterol biosynthesis genes were up-regulated in the shApc-Kras^G12D-Villin-Cre mice colon (n = 4) compared to shRen controls (n = 4) and shApc-Villin-Cre mice (n = 4). M mRNA levels of PCSK9 and cholesterol biosynthesis genes in Apc^Min/+Kras^G12DVillin-Cre mice tumors (n = 6) compared to adjacent normal tissues (n = 5). Data shown are means of biological replicates; ± SEM (C, F–I, K–M). Two-tailed Student’s t-test (C, F, H, I, K–M). Two-tailed two-way ANOVA (G). Source data are provided as a Source Data file.