Fig 2: An HFD compromises the barrier function of the colon. Goblet cell number, tight junctions and cell apoptosis were analyzed to determine the barrier function of the colon. (A) Representative flow cytometry analysis, showing Muc2 expression among the CD326 cell populations. (B) Proportion of goblet cells. (C) Histochemical PAS staining of goblet cells (scale bar, 100 µm; magnification, ×20), (D) percentage of area stained as PAS-positive among the colonic crypts, and (E) quantification of the average number of goblet cells per crypt. (F) mRNA expression of Muc2 in the colon. (G) Representative images of immunofluorescence staining for ZO-1 seen as red fluorescence, with DAPI used as the counterstain (scale bar, 50 µm; magnification, ×40), and (H) quantification of red fluorescence. (I) Representative images of immunofluorescence staining for Claudin-2 seen as red fluorescence, with DAPI used as the counterstain (scale bar, 100 µm; magnification, ×40), and (J) quantification of red fluorescence. (K) mRNA expression of Claudin-2 in the colon. *P<0.05, **P<0.01 and ***P<0.001, vs. Con group. HFD, high-fat diet; Con, control; PAS, Periodic Acid-Schiff; Muc2, mucin 2.

Fig 3: Barrier function of the small intestine was not affected by HFD. Goblet and Paneth cell numbers, and cell apoptosis were analyzed to determine the barrier function of the small intestine. (A) Representative flow cytometry analysis of Muc2 expression in CD326 cell populations. (B) Percentage of goblet cells in the small intestine. (C) Ileal sections were stained for goblet cells using PAS (scale bar, 500 µm; magnification, ×20). (D) Percentage of area stained as PAS-positive among the total villi-crypts sections. (E) Quantification of the number of goblet cells per villus-crypt unit. (F) Representative flow cytometry analysis showing CD24 expression in CD326 cell populations. (G) Percentage of Paneth cells in small intestinal crypts. (H) Crypt-derived organoids of Paneth cells, including lysozyme-positive cells, assessed using immunofluorescence (blue, DAPI; red, lysozyme; scale bar, 200 µm; magnification, ×40). (I) Quantification of Paneth cells in small intestinal crypt-derived organoids. (J) mRNA expression levels of Muc2 in the small intestine of the Con and HFD mice. HFD, high-fat diet; Con, control; PAS, Periodic Acid-Schiff; Muc2, mucin 2.

Fig 4: An HFD increases the ISC count and crypt function in the small intestine of mice. Small intestinal crypts and villi were isolated and epithelial cell suspensions were dissociated into single cells. (A) Representative flow cytometry analysis showing Lgr5 expression in the CD326 cell population obtained from the small intestine. (B) ISCs defined as Lgr5high, and (C) progenitor cells defined as Lgr5low in the entire small intestine. (D) mRNA expression of Lgr5 in the small intestine. (E) Small intestinal crypts were isolated and cultured in Matrigel to develop organoid colonies. Representative images of small intestinal organoids at days 0, 4 and 6 are displayed (scale bar, 100 µm; magnification, ×20). (F) Organoids formed per crypt and (G) crypt-domains per organoid on day 4. (H) Organoids formed per crypt and (I) crypt-domains per organoid on day 6. Data are presented as the mean ± standard deviation. *P<0.05 and ****P<0.0001, vs. Con group. HFD, high-fat diet; ISC, intestinal stem cell; Con, control.

Fig 5: GP38 and SCA1 expression on TMC subsets. (A) Total thymi cells from 1-week-old mice were isolated, and total TMCs (CD45-EpCAM-) were analysed by flow cytometry. t-SNE representation of the expression of CD31, CD140a, CD140ß, GP38, SCA-1, Ly51 and aSMA. (B) Three main clusters were identified: cluster 1 (CD31+), cluster 2 (CD140a+ß+) and cluster 3 (CD140a-ß+). Clusters 2 and 3 were respectively subdivided into cluster 2.1 (CD140a+ß+GP38+SCA1-) and 2.2 (CD140a+ß+GP38+SCA1+); and 3.1 (CD140a-ß+Ly51+aSMA-) and 3.2 (CD140a-ß+Ly51+aSMA+). (C) TMCs (CD45-EpCAM-CD31-) were analysed for the indicated markers, and sub-cluster 2.1 (red gate), 2.2 (green gate), 3.1 and 3.2 (light- and dark-blue gates) were identified. (D) Analysis of GP38, SCA-1 and DPP4 expression in TFA (red gate) and TFB (green gate) populations at E14, E17, 1 week old (W) and 4W. Numbers in plots indicate the frequency of cells found within each gate. Plots are of a representative analysis per time point. (E) Bar graphs showing mean+s.d. of the frequency and cellularity of TFA and TFB subsets, of three independent analyses per time point. Differences in TF subsets, CD140a+ß+GP38+SCA1- (red) and CD140a+ß+GP38+SCA1+ (green), were statistically analysed at different ages: ***P<0.001, **P<0.01, *P<0.05.