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. 2022 Sep 8;219(12):e20212278. doi: 10.1084/jem.20212278

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© 2022 Jimenez et al.

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Figure S2. — miR-181 regulates the IEL milieu at homeostasis. (A and B) Mean percent change in weight from day 0 (A) and colon length (B) of cage-mate miR-181a1-b1^+/−; miR-181a2-b2^+/− (n = 5) and DKO mice (n = 12) treated with 2% DSS starting at 6–8 wk of age. DSS was withdrawn at day 8, and animals were allowed to recover for 2 d. Breeding strategy is illustrated for clarity (A). For colon length (B), each dot represents a single animal. (C) PCA of fecal microbial communities as determined by 16s rRNA-seq of stool from WT (n = 2 per litter) and miR-181a1-b1 KO littermates (n = 2 per litter) from two separate litters. Each dot represents an individual animal. (D) Total number of TCRγδ⁺ and TCRβ⁺ large intestinal IELs from WT and DKO mice at baseline. Each dot represents an individual mouse. Gated on: Singlets CD45⁺ Live (viability⁻) CD103⁺ CD11b⁻ cells. Data pooled from two (WT n = 5, DKO n = 9) independent experiments. (E) Total number of TCR^-, TCRγδ⁺, and TCRβ⁺ large intestinal LP cells from WT and DKO mice at baseline. Each dot represents an individual mouse. Gated on: Singlets CD45⁺ Live (viability⁻) B220⁻ CD11b⁻ cells. Data pooled from two (WT n = 5, DKO n = 9) independent experiments. (F) Total number of TCRβ⁺ CD4^+/− CD8β^+/− large intestinal LP cells from WT and DKO mice at baseline. Each dot represents an individual mouse. Gated on: Singlets CD45⁺ Live (viability⁻) B220⁻ CD11b⁻ TCRβ⁺ LP cells. Data pooled from two (WT n = 5, DKO n = 9) independent experiments. (G) Total number of large intestinal LPMs: monocytes (Ly-6C⁺ Ly-6G⁻, monos), neutrophils (Ly-6C⁻ Ly-6G⁺, neuts), eosinophils (Ly-6C⁻ Ly-6G⁻ Siglec-F⁺ SSC-A^hi, eos), CD11b⁺ dendritic cells (Ly-6C⁻ Ly-6G⁻ Siglec-F^- MHCII⁺ CD64⁻ CD11c⁺, DCs), and macrophages (Ly-6C⁻ Ly-6G⁻ Siglec-F⁻ MHCII⁺ CD11c^+/− CD64⁺, macs) in WT and DKO mice at baseline. Gated on: Singlets CD45⁺ Live (viability⁻) B220⁻ intracellular CD3ε⁻ CD11b⁺ cells. Each dot represents a single mouse. Data pooled from two (WT n = 5, DKO n = 9) independent experiments. (H and I) Percentage (H) and total number (I) of TCRγδ⁺ large intestinal IELs expressing CD8α from WT (n = 8–9) and DKO (n = 12) mice after 5 d of 2% DSS treatment. Each dot represents an individual mouse. Gated on: Singlets CD45⁺ Live CD103⁺ CD11b⁻ TCRγδ⁺ cells. Data pooled from two independent experiments. (J) Percentage of TCR⁻, TCRγδ⁺, and TCRβ⁺ large intestinal LPLs from WT (n = 8–9) and DKO (n = 12) mice after 5 d of 2% DSS treatment. Each dot represents an individual mouse. Gated on: Singlets CD45⁺ Live CD103⁺ CD11b⁻ cells. Data pooled from two independent experiments. (K) Total number of TCRβ⁺ large intestinal LPLs expressing CD4 and CD8β from WT (n = 8–9) and DKO (n = 12) mice after 5 d of 2% DSS treatment. Each dot represents an individual mouse. Gated on: Singlets CD45⁺ Live CD103⁺ CD11b⁻ TCRβ⁺ cells. Representative FACS plot shown on the right. Data pooled from two independent experiments. (L and M) Percentage (L) and total number (M) of TCRγδ⁺ large intestinal IELs expressing CD8α from WT (n = 3) and DKO (n = 3) mice after 7 d of 2% DSS treatment and 7 d of recovery. Each dot represents an individual mouse. Gated on: Singlets CD45⁺ Live CD103⁺ CD11b⁻ TCRγδ⁺ cells. (N) Percentage of TCR⁻, TCRγδ⁺, and TCRβ⁺ large intestinal LPLs from WT (n = 3) and DKO (n = 3) mice after 7 d of 2% DSS treatment and 7 d of recovery. Each dot represents an individual mouse. Gated on: Singlets CD45⁺ Live CD103⁺ CD11b⁻ cells. (O) Total number of TCRβ⁺ large intestinal LPLs expressing CD4 and CD8β from WT (n = 3) and DKO (n = 3) mice after 7 d of 2% DSS treatment and 7 d of recovery. Each dot represents an individual mouse. Gated on: Singlets CD45⁺ Live CD103⁺ CD11b⁻ TCRβ⁺ cells. Representative FACS plot shown on the right. Error bars indicate mean ± SEM. Two-way ANOVA with Sidak’s multiple comparisons test (A), Student’s unpaired t test (B), multiple t tests with FDR correction (D–O). *, P < 0.05; ***, P < 0.001. Each dot represents a single mouse (B–O). Significance values for paired genotype comparisons from multivariate analyses are indicated with a vertical bar and appropriate P- or q-value (represented by asterisk) when appropriate. Significance values for multiple comparisons tests assessing day-to-day changes in weight loss between groups are indicated by P values next to individual data points if <0.05.