Additional file 2 of Gut microbiota regulation of P-glycoprotein in the intestinal epithelium in maintenance of homeostasis
Creators: name: Foley, Sage E.
name: Tuohy, Christine
name: Dunford, Merran
name: Grey, Michael J.
name: De Luca, Heidi
name: Cawley, Caitlin
name: Szabady, Rose L.
name: Maldonado-Contreras, Ana
name: Houghton, Jean Marie
name: Ward, Doyle V.
name: Mrsny, Randall J.
name: McCormick, Beth A.
Publisher: name: figshare
Year: 2021
Subjects: subject: Biochemistry
subject: Physiology
subject: FOS: Biological sciences
subject: 39999 Chemical Sciences not elsewhere classified
subject: FOS: Chemical sciences
subject: Science Policy
subject: Immunology
subject: FOS: Clinical medicine
subject: Cancer
subject: 110309 Infectious Diseases
subject: FOS: Health sciences

description: Additional file 1: Figure S1. Intestinal bacterial load and P-gp expression are both reduced within 5 days of antibiotic treatment. A WT SPF mice were treated with AVNM cocktail for 5 days. One group of mice were euthanized on each day. A representative blot of P-gp protein expression in colonic tissue is shown, each lane representing a replicate mouse within each group. An “internal control” (IC) lysate was included across multiple blots for data comparison. B Densitometry data are shown for experiments performed in (A), pooled from two independent experiments, N = 11 mice per group. ***p = 0.0001, ****p &lt; 0.0001, one-way ANOVA with Dunnett’s multiple comparisons test. C Fold differences in relative amount of 16S DNA in feces collected from mice on Days 0-5 of AVNM delivery are shown for data pooled from two independent experiments, N = 11 mice per group. ****p &lt; 0.0001, one-way ANOVA with Dunnett’s multiple comparisons test, based on ΔΔ Ct values. D Fold change differences in relative 16S DNA data are shown from C overlaid with relative P-gp protein expression from densitometry B, with data representing the mean. Figure S2. Individual antibiotic treatment does not affect P-gp expression directly or through epithelial loss. A WT SPF mice were treated with ampicillin for 10 days, as in Fig. 1. Fold difference in relative amount of 16S DNA in feces collected from mice on Day 10 of antibiotic delivery, relative to control. Data from two independent experiments are shown individually with ampicillin treatment compared to control. N = 5 mice per group per experiment; ****p &lt; 0.0001, two-way ANOVA with Tukey’s multiple comparisons test. B Representative western blot showing Villin expression in colonic tissue from mice treated with antibiotics as in A and Fig. 1, each lane representing a replicate mouse within each group. Densitometry calculated relative to control group; N = 3 per group, ns p &gt; 0.05, one-way ANOVA. C Representative western blot showing P-gp expression in T84 lysates after incubation with antibiotic for 24 hrs. Densitometry calculated relative to media control samples. Data are pooled from two independent experiments; ns p &gt; 0.05, one-way ANOVA. Figure S3. Short-chain fatty acids and secondary bile acids induce P-gp expression without affecting toxicity and TER. A Representative western blot of P-gp expression in T84 cells incubated with acetate, propionate, butyrate, or a combination of the three short-chain fatty acids (SCFAs) at a range of concentrations, all in physiologically relevant ratios. B Densitometry data for samples pooled from two independent experiments are compared to media control lysate; *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001, one-way ANOVA with Tukey’s multiple comparisons test. C LDH release into supernatants from T84 cells incubated with butyrate and/or bile acids at the indicated concentrations for 24 hrs are shown. 5% DMSO was included as a positive control for toxicity. Data are samples pooled from two independent experiments; ns p &gt; 0.05, one-way ANOVA. D Percent change in TER measured in transwell plates of T84 cells before and after 24hr incubation with butyrate and/or bile acids at the indicated concentrations. 5% DMSO was included as a control for toxicity and/or TER reduction. Data are shown for samples pooled from two independent experiments; ns p &gt; 0.05, *p &lt; 0.05, one-way ANOVA with Tukey’s multiple comparisons test. Figure S4. Bile acid sequestration in the intestine. Bile acid content of intestinal tissue from colon or cecum after delivery of cholestyramine (CME) in the chow for 14 days. Data shown are pooled from two independent experiments; N = 7-8 per group; **p &lt; 0.01, ***p &lt; 0.0001 for unpaired t-test. Figure S5. P-gp expression is decreased specifically with increasing inflammation in UC patients. A Representative western blot of villin expression in human patient colon biopsies, as in Fig. 5. B Densitometry data for relative villin expression compared to that of P-gp, color coded by patient sample type – healthy control, UC uninvolved, UC involved. There appears to be a correlation between P-gp expression and villin expression (left), however significance is lost when pan-colitis patients with severe inflammation and presumed wholesale epithelial damage are removed (right). Table S1. Spearman correlation results of bacterial genera and P-gp. Shown are p-values and rho coefficients from Spearman correlation tests of each bacterial genus identified through whole genome sequencing aligned with relative colonic P-gp expression data generated from western blot densitometry. Table S2. Human patient metadata. Shown are sex, age, diagnosis, and fecal calprotectin levels for each human patient from which samples were tested for P-gp expression in biopsies (Fig. 5B,C), endocannabinoid content in mucosal brushings (Fig. 5D) or P-gp induction by fecal samples (Fig. 5E). Patients were binned by calprotectin levels determined by ELISA into three groups – high inflammation, low inflammation, and no inflammation.