Fig. 2: NAFLD promotes the stemness properties of liver metastatic CRC cells.

A Representative immunofluorescence images for CD44 and CD133 (red) expression of MC38 allografts in control and NAFLD mice with intrahepatic implantation. DAPI (blue) to mark nucleus. Scale bars, 20 μm. Right, quantification of the results (n = 5). B Representative IHC staining images for Oct4, Nanog and SOX2 of MC38 allografts in control and NAFLD mice with intrahepatic implantation. Scale bars, 50 μm. Below, quantification of the results (n = 5). C Sphere formation assay on tumor cells isolated from HCT 116 xenografts in control and NAFLD mice. Representative photos of HCT 116 sphere in the second passage were taken on day 10 after cells were seeded (scale bars, 200 μm), and sphere numbers and diameters were determined and plotted (n = 3). D The protein level of CSC markers Oct4, Nanog and SOX2 of HCT 116 xenografts in control and NAFLD mice by western blot assay (n = 5). E Sphere formation assay on HCT 116 and SW 480 cells treated with BSA-PA/OA at indicated concentrations (PA 0 µM, OA 0 µM; PA 50 µM, OA 100 µM; PA 100 µM, OA 200 µM; PA 200 µM, OA 400 µM, respectively). Representative photos of sphere in the second passage were taken on day 10 after cells were seeded (scale bars, 100 μm), and sphere numbers and diameters were determined and plotted (n = 3). F Protein level of CSC markers of HCT 116 and SW 480 cells treated with BSA-PA/OA at different concentrations (PA 0 µM, OA 0 µM; PA 50 µM, OA 100 µM; PA 100 µM, OA 200 µM; PA 200 µM, OA 400 µM, respectively) detected by western blot assay. G In vitro limiting dilution analysis for frequency of CSCs in HCT 116 cells treated with BSA or BSA-PA/OA, calculated with Extreme Limiting Dilution Analysis software (http://bioinf.wehi.edu.au/software/elda/). Data are presented as mean ± SEM. Significance was determined by two-tailed unpaired Student’s t test (A–C), one-way ANOVA (E) and Pairwise test (G), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. NAFLD non-alcoholic fatty liver disease, BSA bovine serum albumin, PA palmitic acid, OA oleic acid.