Supplementary Figures 1 - 16, Tables 1 - 3 from A Gnotobiotic Mouse Model Demonstrates That Dietary Fiber Protects against Colorectal Tumorigenesis in a Microbiota- and Butyrate-Dependent Manner

Abstract

<p>Supplementary Figure 1. Overall experimental design including the rationale behind specific details. Supplementary Figure 2. Confirmation of the ASF strains and B. fibrisolvens by PCR. Supplementary Figure 3. Formulations of the 3 diets provided to gnotobiotic mice in these experiments. Supplementary Figure 4. A combination of a high-fiber diet and a butyrate-producing bacterium (Butyrivibrio fibrisolvens) yields higher levels of butyrate in the lumen of the colon than either factor in isolation. Supplementary Figure 5. Detection of b-catenin mutations in tumor cells. Supplementary Figure 6. Analysis of b catenin nuclear localization in control and experimental tumors. Supplementary Figure 7. Characterization of squamous cell tumors. Supplementary Figure 8. Analysis of wild-type and mutant B. fibrisolvens strains. Supplementary Figure 9. Expression of Warburg effect markers. Supplementary Figure 10. Butyrate oxidation and uptake in colorectal tumors and normal colonic tissues. Supplementary Figure 11. Representative IHC images showing cleaved caspase 3 positive cells (arrows) in normal colonic tissue and tumors from mice in control and experimental conditions. Supplementary Figure 12. Flow cytometric analysis of immune cell populations from BALB/c mice colonized with the 4 ASF and B. fibrisolvens in gnotobiotic isolators. Supplementary Figure 13. Serum and colonic cytokine levels (pg/mL) from BALB/c mice colonized with the 4 ASF and B. fibrisolvens in gnotobiotic isolators based on Luminex assays. Supplementary Figure 14. Minimal inflammation based on histologic assessment of H&E-stained sections of colorectal tumor sections from 7-10 mice of each treatment group 5 months after exposure to the 5 AOM/3 DSS regimen. Supplementary Figure 15. Control experiment demonstrating that inflammation plays a minor role in colorectal tumorigenesis in the gnotobiotic model where BALB/c mice were polyassociated with 4 ASF plus B. fibrisolvens and then treated with AOM/DSS. Supplementary Figure 16. Relative Il18 mRNA levels normalized to Gapdh levels in normal colonic tissue and tumors based on RT-qPCR experiments. Supplementary Table 1. Luminal Concentrations (μM) of Other Two Major SCFAs (Propionate and Acetate). Supplementary Table 2. Clinical Information Regarding Human Adenocarcinomas. Supplementary Table 3. Statistical tests used for each figure panel.</p>