Supplementary Figures S1-S3
Supplementary Figure 1. Cecum GABA concentration, pH, and primer sequences: A.) Cecal digesta pH (n=30 per diet). B.) GABA concentrations of cecal contents on a dry matter basis (n=30 per diet)
Supplementary Figure 2. Sex differences in ileum related outcomes: A.) γ-aminobutyric acid (GABA) concentrations of ileum contents on a dry matter basis in male vs female mice (n=16 per bar in males, n=14 per bar in females). B.) Volcano plot depicting differential expression of intestinal motility-related gene expression of whole-ileum tissue between sexes with significance (-log10 p-value) on the y-axis and effect size (log2-fold difference) on the x-axis. The y-axis threshold represented as the horizontal grey dotted line was set at p < 0.05 (1.3 –log10p-value) to signify significant differences in gene expression by the main effect of sex using female mice as a reference group. Genes positioned to the left of the vertical left grey line threshold indicate downregulated expression of a particular gene in males compared to females. Genes positioned to the right of the vertical left grey line threshold indicate upregulated expression of a particular gene in males compared to females. The grey line threshold was set at a 0.08 log2 fold difference to plot all genes differentially expressed by sex, based on independent two-sample t-tests after meeting assumptions. Colored magenta dots represent significantly differentially expressed genes. Black dots represent genes that are not differentially expressed between males and females. (N=120) C.) Fiber-free diet (FFD) vs Cellulose diet (CELL) comparison of ileal gene expression in male mice (n=16 FFD, n=16 CELL) D.) FFD vs CELL comparison of ileal gene expression in female mice (n=14 FFD, n=14 CELL) E.) FFD vs short-chain fructooligosaccharide diet (scFOS) comparison of ileal gene expression in male mice (n=16 FFD, n=16 scFOS) F.) FFD vs scFOS comparison of ileal gene expression in female mice (n=14 FFD. N=14 scFOS) G.) FFD vs Inulin (INU) comparison of ileal gene expression in male mice (n=16 FFD n=16 INU) H.) FFD vs INU comparison of ileal gene expression in female mice (n=14 FFD, n=14 INU) I.) Visual illustration of ileal ex-vivo motility assay J.) Sex-dependent differences in contraction force measured as total area under the curve of initial contraction measured in 2-cm segments of ileum tissue post-acetylcholine stimulation (n=12 per bar). K.) Sex-dependent differences in contraction duration of initial contraction measured in 2-cm segments of ileum tissue post-acetylcholine stimulation (n=13 per bar). L.) Sex-dependent differences in contraction power (force/time) of initial contraction measured in 2-cm segments in ileum tissue post-acetylcholine stimulation (n= 12 per bar).
Supplementary Figure 3. Sex differences in proximal colon related outcomes: A.) γ-aminobutyric acid (GABA) concentrations of proximal colon contents on a dry matter basis in male vs female mice (n=16 per bar in males, n=14 per bar in females). B.) Volcano plot depicting differential expression of intestinal motility-related gene expression of whole-proximal colon tissue between sexes with significance (-log10 p-value) on the y-axis and effect size (log2-fold difference) on the x-axis. The y-axis threshold represented as the horizontal grey dotted line was set at p < 0.05 (1.3 –log10p-value) to signify significant differences in gene expression by the main effect of sex using female mice as a reference group. Genes positioned to the left of the vertical left grey line threshold indicate downregulated expression of a particular gene in males compared to females. Genes positioned to the right of the vertical left grey line threshold indicate upregulated expression of a particular gene in males compared to females. The grey line threshold was set at a 0.08 log2 fold difference to plot all genes differentially expressed by sex, based on independent two-sample t-tests after meeting assumptions. Colored magenta dots represent significantly differentially expressed genes. Black dots represent genes that are not differentially expressed between males and females. (N=120) C.) Fiber-free diet (FFD) vs Cellulose diet (CELL) comparison of colonic gene expression in male mice (n=16 FFD, n=16 CELL) D.) FFD vs CELL comparison of colonic gene expression in female mice (n=14 FFD, n=14 CELL) E.) FFD vs short-chain fructooligosaccharide diet (scFOS) comparison of colonic gene expression in male mice (n=16 FFD, n=16 scFOS) F.) FFD vs scFOS comparison of colonic gene expression in female mice (n=14 FFD. N=14 scFOS) G.) FFD vs Inulin (INU) comparison of colonic gene expression in male mice (n=16 FFD n=16 INU) H.) FFD vs INU comparison of colonic gene expression in female mice (n=14 FFD, n=14 INU) I.) Visual illustration of proximal colon ex-vivo motility assay J.) Sex-dependent differences in contraction force measured as total area under the curve of initial contraction measured in 2-cm segments of proximal colon tissue post-acetylcholine stimulation (n=12 per bar). K.) Sex-dependent differences in contraction duration of initial contraction measured in 2-cm segments of proximal colon tissue post-acetylcholine stimulation (n=13 per bar). L.) Sex-dependent differences in contraction power (force/time) of initial contraction measured in 2-cm segments in proximal colon tissue post-acetylcholine stimulation (n= 12 per par).
