Fig. 7

BHB-treated M2 macrophages promote intestinal epithelial cell proliferation. a, b Littermate WT mice received BHB or saline enema were given DSS in drinking water to induce experimental colitis as described in Fig. 2a. On day 18, these mice were killed and their colon tissues were collected for IHC analyses. a Representative IHC images of Brdu and PCNA immunostaining in the colon tissues (scale bars: 150 μm). b Quantitative analyses of Brdu and PCNA staining by Image-Pro-Plus software. Data represent means ± SEM from 6 mice in each group. c–e BMDMs isolated from mice were stimulated with IL-4, and then treated with BHB or AS1517499 alone or together. Forty-eight hours later, the cell culture supernatant purified by ultrafiltration was used to stimulate IEC-6 cells (c), and the cells were co-cultured with colon organoids in a transwell system (d, e). c IEC-6 cell viability was measured by CCK-8 assay. Data represent means ± SD from quintuplicate wells in each group. d Colon organoids were harvested and counted after 9-day supernatant stimulation. Data represent means ± SEM from three independent experiments. e Representative images of colon organoids after 3-, 6-, and 9-day supernatant stimulation (scale bars: 300 μm). f Representative images of colon organoids after 9-day supernatant stimulation (scale bars: 50 μm).g Quantitative analyses of average size of (day 9) colonic organoids. h Quantitative analyses of new crypt formation (budding) of (day 9) organoids. NS, not significant; *P < 0.05, **P < 0.01 by unpaired Student’s t test. Data shown are representative of three (a–c) independent experiments