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Additional file 9: Figure S9. The effect of RhoB on intestinal phenotype depends on its expression in epithelia. CD45.1 WT bone marrow were transferred into irradiated CD45.2 WT, RhoB+/- or RhoB-/- mice respectively (n = 6 from 2 independent experiments) (A) Scheme of bone marrow transplantation protocol. (B) Flow cytometry analysis of CD45.1 expression in peripheral blood cells after bone marrow transplantation. The percentage of CD45.1+cells in lymphocyte cells from the indicated genotypes. (C) Representative H&E staining analysis of histopathological changes in colon of the indicated genotypes. (D) Representative AB-PAS staining and quantification in colon sections of the indicated genotypes. (E) Representative confocal images and quantitation of Muc2 staining (green) and DAPI (blue) in colonic tissues of the indicated genotypes. (F) Ki67 staining in colon sections of the indicated genotypes. (G) Representative GPR41 and GPR43 staining and quantitation in colon sections as indicated. Scale bar: 50 μm or 10 μm. Data are the mean ± SD. One-way ANOVA (B-G). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Additional file 5: Figure S5. Altered microbiota in RhoB+/- and RhoB-/- mice. Stool samples from 6-8 week old WT, RhoB+/- or RhoB-/- mice were collected and analyzed by 16S rRNA gene sequencing (n = 5). (A) The amounts of bacterial DNA. (B) Analysis of the Shannon diversity index of microbiota. (C) PCoA results based on unweighted UniFrac distances. (D) Relative abundance of bacteria by taxon-based analyses. (E) Heatmap to visualize the relative abundances of the 30 most predominant bacterial genera.

Additional file 2: Figure S2. RhoB deficiency alleviates inflammatory responses to DSS treatment. (A) mRNA expression levels of anti-microbial peptides and proinflammatory cytokines in the colons of indicated mice measured by real-time PCR (n = 9 from 2 independent experiments). (B) Gating strategy for flow cytometry analysis of immune cells. (C) Representative flow cytometry analysis of the indicated cells (left) and percentage (right) of indicated cells in mesenteric lymph nodes (MLN) and colonic lamina propria (cLP) from indicated naive mice (n = 9 from 2 independent experiments). (D) Representative flow cytometry analysis of the indicated cells (left) and percentage (right) of indicated cells in MLN and cLP from indicated mice after DSS administration (n = 9 from 2 independent experiments). Data are the mean ± SD. Statistical significance was determined by one-way ANOVA (A, C and D). *p < 0.05, **p < 0.01, ***p < 0.001.

Additional file 3: Figure S3. RhoB deficiency protects mice from DSS-induced chronic colitis. (A-F) Eight-week-old male mice were given 1% DSS in drinking water for seven days and followed by normal drinking water for an additional 7 days with 3 cycles of treatment (n = 9 from 2 independent experiments). (A) Schematic image illustrating chronic colitis design. (B) Body weight loss of the indicated mice and treatments. (C) Disease activity index of the indicated mice and treatments. (D) Measurement and quantification of colon length in the indicated mice. (E) Representative H&E staining analysis of histopathological changes and quantitation of histology score in colon from the indicated mice. (F) Alcian blue-Periodic acid Schiff (AB-PAS; indicating goblet cells) staining in colon from the indicated mice and treatments. Scale bar: 50 μm. (G) Measurement and quantification of colon length in naïve WT, RhoB+/-, and RhoB-/- mice (n = 9 from 3 independent experiments). (H) Representative H&E staining analysis of histopathological changes in colon from naïve WT, RhoB+/- and RhoB-/- mice. (I) Representative Lgr5, Occludin, ZO-1, and cleaved caspase-3 staining and quantitation in colon sections as indicated (n = 9 from 3 independent experiments). Scale bar: 50 μm. Data are the mean ± SD. Statistical significance was determined by two-way ANOVA (B and C) or Unpaired Student’s t-test (D-F) or one-way ANOVA (G and I). **p < 0.01, ***p < 0.001, ****p < 0.0001. NS, not significant.

Additional file 11: Figure S11. Autophagy does not affect Ki67, GPR41 and GPR43 protein levels. Mice were treated with rapamycin for 7 days and then were analyzed (n = 8 from 2 independent experiments). (A) Representative AB-PAS staining and quantification in colonic tissues of the indicated genotypes. (B) Representative Muc2 staining and quantitation in colon sections as indicated. (C) Representative confocal images of Muc2 staining in colonic tissues of the indicated genotypes. Quantification of the inner mucus layer thickness measured from the top of the villi to the periphery of the Muc2-positive region. Muc2: green; DAPI: blue. (D) Representative Ki67 staining and quantitation in colon sections as indicated. (E) Representative GPR41 staining and quantitation in colon sections as indicated. (F) Representative GPR43 staining and quantitation in colon sections as indicated. Scale bar: 10, 50 or 200 μm. Data are the mean ± SD. One-way ANOVA. ****p < 0.0001. NS, not significant.

Additional file 6: Figure S6. SCFA concentrations are positively correlated to the relative abundance of Prevotella and Alloprevotella. (A) Pearson’s correlation between the relative abundance of Prevotella and SCFAs in fecal from WT, RhoB+/-, and RhoB-/- mice (n = 15). (B) Pearson’s correlation between the relative abundance of Alloprevotella and SCFAs in fecal from WT, RhoB+/-, and RhoB-/- mice (n = 15). (C-F) Abx treatment eliminates the difference in the colitis phenotype of WT and RhoB-deficient mice (n = 5). (C) Scheme of Abx treatment protocol. (D) Disease activity index of WT and RhoB+/- mice. (E) Measurement and quantification of colon length in WT and RhoB+/- mice. (F) Representative H&E staining analysis of histopathological changes and quantitation of histology score in colons of WT and RhoB+/- mice. Boxed areas in colon sections are enlarged and shown at the bottom panel. Scale bar: 200 μm or 50 μm. Data are the mean ± SD. Significance is determined using linear regression (A-B) or two-way ANOVA (D) or unpaired Student’s t-test (E and F). NS, not significant.

Additional file 7: Figure S7. The microbiota composition in the feces from WT mice shifts toward that of RhoB-deficient mice after cohousing. Stool samples from WT and RhoB-deficient mice before and after cohousing were collected and analyzed by 16S rRNA gene sequencing (n = 4). (A) Analysis of the Shannon diversity index of microbiota in the indicated genotypes. (B) PCoA analysis of microbiota. (C) Relative abundance of fecal microbiota at the genus level. (D) LEfSe analysis of distinctive microbiota composition as indicated.

Additional file 8: Figure S8. Intestinal microbiota does not affect goblet cell numbers, Muc2 and Ki67 protein levels. (A-C) WT and RhoB-/- mice were cohoused for 4 weeks (n = 8 from 2 independent experiments). (A) Representative GPR41 and GPR43 staining and quantitation in colon sections as indicated. Scale bar: 50 μm. (B) Representative AB-PAS staining and quantification in colon sections of the indicated genotypes. (C) Representative Muc2 and Ki67 staining and quantitation in colon sections as indicated. (D-F) Autophagy induced by rapamycin does not reverse the colitis phenotype in RhoB-/- mice. Mice were treated with rapamycin for 7 days and then treated with 1.5% DSS (n = 4 or 5). (D) Disease activity index of the indicated genotypes. (E) Measurement and quantification of colon length in the indicated genotypes. (F) Histopathological changes and quantitation of histology score in colon of the indicated genotypes. Scale bar: 50 μm. Data are the mean ± SD. Unpaired Student’s t-test (A to C) or two-way ANOVA (D) or one-way ANOVA (E-F). *p < 0.05, **p < 0.01, ***p < 0.001. NS, not significant.