Fig 1: CS promotes cholesterol biosynthesis by binding to NPC2 and activating SREBP2.a Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways analysis for the up-regulated differentially expressed genes in CS-treated HT-29 cells compared with control HT-29 cells (treated with DMSO). (Fold change =1.5, P < 0.05). b Heatmaps for the steroid biosynthesis genes in the upregulated genes group in CS-treated HT-29 cells compared with control HT-29 cells (treated with DMSO). c RT-qPCR analysis of HMGCS1, DHCR7, FDFT1 and CYP51A1 mRNA levels in HT-29 cells, LOVO cells, SW480 cells, HCT116 cells, SW1116 cells, and NCM460 cells treated with CS (50 µM) or DMSO for 24 h (n = 2 independent culture wells, two-tailed Student’s t-test). d The cholesterol concentration of HT-29 cells treated with CS (50 µM) for 6, 12, 24, and 48 h measured by the Amplex® Red Cholesterol Assay Kit (n = 4 biologically independent samples, one-way ANOVA with Sidak’s multiple comparison test). e Filipin III staining in HT-29 and SULT2B1-KO HT-29 cells in the presence or absence of CS (50 µM) (n = 4 fields/group, one-way ANOVA with Sidak’s multiple comparison test). f Cell viability of HT-29 and SULT2B1-KO cells treated with 25 µM or 50 µM CS (n = 4, 3, 3 biologically independent samples/group, respectively, one-way ANOVA with Sidak’s multiple comparison test). g Transcriptional profiles of SLC family members in colonic tissues from Sult2b1f/f and Sult2b1?IEC mice challenged with 2.5% DSS (n = 4 mice/group). h Normalized FPKM of Slc10a6 in inflamed colonic mucosa based on RNA-seq data of Sult2b1f/f and Sult2b1?IEC mice with colitis (n = 4 mice/group, two-tailed Student’s t test). i Immunofluorescent staining of SLC10A6 in SULT2B1-KO + Ad-GFP and SULT2B1-KO + Ad-SLC10A6 cells, where nuclei were counterstained with DAPI. (n = 2 biologically independent samples). j Western blotting analysis of SLC10A6-Flag in SULT2B1-KO + Ad-GFP and SULT2B1-KO + Ad-SLC10A6 cells. (n = 4 biologically independent samples). k The luciferase activity was measured in SULT2B1-KO and SULT2B1-KO + Ad-SLC10A6 cells treated with DMSO, and 5 µM, 10 µM, 25 µM, 50 µM, and 100 µM CS, and the results were normalized, with respect to measurements corresponding to DMSO treatment (n = 2 biologically independent samples, one-way ANOVA with Sidak’s multiple comparison test). l Cellular thermal shift assay of HT-29 cells in the presence or absence of 50 µM CS. Two-way ANOVA with Tukey’s multiple comparisons test, 57 °C (DMSO vs. CS) *P = 0.0316; 60 °C (DMSO vs. CS) **P = 0.0076; 63 °C (DMSO vs. CS) **P = 0.0063; 66 °C (DMSO vs. CS) **P = 0.0044. Data from in vitro assays are reprehensive of at least three independent experiments. Data are shown as the mean ± SEM. Source data are provided as a Source Data file.
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