Fig 2: Myeloid-specific deletion of Irf5 inhibits intestinal epithelial cell apoptosis in experimental NEC. a Representative TUNEL (red) staining of the intestines of the indicated groups. Intestinal epithelial cells were stained with EpCAM (green). NEC significantly increased TUNEL+EpCAM+ cells in the Irf5fl/fl pups, while Irf5 deletion in myeloid cells markedly blocked this effect. b Western blotting analysis of cleaved caspase-3 expression in the intestines of the indicated groups. The expression of cleaved caspase-3 was significantly increased in the Irf5fl/fl pups with NEC compared to the breast-fed Irf5fl/fl pups, while Irf5 deletion in myeloid cells significantly suppressed this effect. (n = 5 in the breast-fed Irf5fl/fl group; n = 5 in the breast-fed Irf5ΔMΦ group; n = 13 in the NEC Irf5fl/fl group; n = 12 in the NEC Irf5ΔMΦ group. Scale bar: 100 μm. *P < 0.05)

Fig 3: Hedgehog-responsive cells in the intestine are fibroblast-like cells. (A) ZsGreen expression in small intestine and colon of Gli1-CreERT2-Rosa26-ZsGreen animals upon induction with tamoxifen. Nuclei were visualized by 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bars: 50 μm. (B) Flow cytometry for expression of Epcam, CD45, CD31, and gp38 in ZsGreen+ and ZsGreen- cells of induced Gli1-CreERT2-Rosa26-ZsGreen animals. Blue lines indicate isotype control, red line indicates specific staining. (C) Immunofluorescent staining for gp38, collagen III, platelet-derived growth factor receptor-α (PDGFRα), CD45, and Lyve1 (magenta) on sections generated from induced Gli1-CreERT2-Rosa26-ZsGreen animals. Nuclei were visualized by DAPI (blue). Arrows indicate co-localization. Scale bars: 25 μm; scale bars in overview, 50 μm. (D) Quantitative RT-PCR for Hedgehog targets Gli1 and Ptch1 in intestinal cell populations positive for Epcam, CD45, CD31, or gp38, respectively, isolated from wild-type mice (N = 3).

Fig 4: Effects of glyceollin I and II on epithelial duct growth in mouse mammary glands and on ER-positive and ER-negative breast cell lines. Mammary glands were obtained from non-ovariectomized (non-ovx) or ovariectomized (ovx) mice treated with vehicle, 10 μg/kg E2, 50 mg/kg glyceollin I or glyceollin II (GI L or GII L, respectively), or 100 mg/kg glyceollin I or glyceollin II (GI H or GII H, respectively) either alone or in combination with E2 for 72 h (a). Epithelial duct length was assessed by delimiting an area around the lymphatic ganglion and measuring the length of this area using FIJI software (Cartridge). The results are represented in box-and-whisker plots, where the top and the bottom of the box correspond to the 75th and the 25th percentile, respectively. The horizontal bar in the box is the median, and the points outside the box correspond to extreme values. The results are expressed in μm/μm2 +/− SEM and were obtained from four independent experiments with at least 5 mice per group. *p-value <0.05 and ***p-value <0.001 with a Mann-Whitney test followed by Bonferroni correction for comparisons of ovx vs treated mice. #p-value <0.05, ##p-value <0.01 and ###p-value <0.001 with a Mann-Whitney test followed by Bonferroni correction for comparisons of E2 vs the other treatments. The anti-proliferative effect of glyceollin was assessed using Ki-67 and Epcam immunostaining of mammary gland tissue (b and cartridge). The immunostaining was performed on frozen slices of mammary glands around the lymphatic ganglion for ovx and E2-treated mice in combination with the two glyceollin concentrations. Then, the number of Ki-67-positive epithelial cells was determined with FIJI software. The results are expressed as the percentage of Ki-67-positive epithelial cells and represent the mean +/− SEM of three independent mice per treatment. ***p-value <0.001 with a Mann-Whitney test followed by Bonferroni correction for comparisons of ovx vs treated mice. ###p-value <0.001 with a Mann-Whitney test followed by Bonferroni correction for comparisons of E2 vs the other treatments. The effects of glyceollin on cell proliferation were also assessed in the ER-positive breast cell lines MCF-7 (c), T47D (d), and HC-11 (e) and the ER-negative breast cell line MCF10A (f). Cells were grown for 6 days with or without 10−9 M estradiol (E2) in the presence or absence of different concentrations of glyceollins. The numbers of cells measured in the presence of glyceollin I (black hatched line) or II (grey hatched line), either alone or in combination with E2 (full line), are expressed as percentages of E2-treated cells (c and d) or untreated control cells (e and f). The results are presented as the mean +/− SEM of four independent experiments

Fig 5: Adipocyte exosomes reduce the sensitivity of CRC organoids to L‐OHP: A) Schematic description of the organoids development. B) Representative images of organoids growth were recorded by bright‐field microscope. Scale bar = 100 µm. C) Multiplex Fluorescent Immunohistochemistry Staining images of organoids expressing specific markers CD44 (red), Ki67 (pink), CEA (yellow) and EP‐CAM (green) with blue DAPI staining (scale bar = 500 µm). D) Immunohistochemistry analysis of CD44, Ki67, CEA and EP‐CAM levels in serial organoids sections (scale bar = 500 µm). E) Cell viability assay detection of the viability of oxaliplatin‐exposed organoids. F) Representative images of organoids treated with L‐OHP, pretreated with MTTP siRNA, exosomes secreted by KD‐MTTP adipocytes or mature adipocyte exosomes and then treated with oxaliplatin (scale bar = 200 µm). G) Cell viability of organoids described in (F). Data are presented as means ± SD of three simultaneously performed experiments (G). P value was calculated using one‐way ANOVA; n.s., not significant; *P < 0.05, **P < 0.01, ***P < 0.001.