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Necropsy of APC Min/+ mice Adenoma in small intestine Spleen 8-10 weeks 18-25 weeks + + - + Normal spleen architecture Expansion of erythroid progenitors.

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Presentation on theme: "Necropsy of APC Min/+ mice Adenoma in small intestine Spleen 8-10 weeks 18-25 weeks + + - + Normal spleen architecture Expansion of erythroid progenitors."— Presentation transcript:

1 Necropsy of APC Min/+ mice Adenoma in small intestine Spleen 8-10 weeks weeks Normal spleen architecture Expansion of erythroid progenitors Adenoma in large intestine Disrupted epithelial lining - + Supplementary figure 1 Colonic tumors and anemia manifest at an older age of APC Min/+ mice. Necropsy was performed on SPF APC Min/+ mice of different age groups, as indicated. Tumor scoring and characterization of epithelial lining disruption were performed on paraffin embedded tissue sections.

2 APC Min/+ mice display disrupted epithelial lining in colonic tumor regions and have increased intestinal barrier permeability. Colon and small intestine samples from weeks old APC Min/+ mice were fixed, sectioned and stained with H&E. Picture represents cross-section of tumors in small intestine and colon, as indicated. Pictures in lower panels present magnified frames of the panels above them. The arrows in upper panels indicate sites with damaged epithelial lining. Supplementary figure 2 Small Intestine Colon Infiltrating cells

3 Small intestine Colon CD11b GR1 Supplementary figure 3 T T T T APC Min/+ mice display increased myeloid cell recruitment in colonic tumors compared to small intestinal tumors. Tissues from colon and small intestine of weeks old APC Min/+ mice were snap-frozen, cryostat sectioned and immunofluorescent staining was performed. Picture represents cross- section of tumors in small intestine and colon. Tumor regions are marked by dashed lines and labeled as ‘T’. Upper and lower panels show immunofluorescent staining of myeloid cell markers CD11b and GR1 (green) respectively while cell nuclei are counterstained with dapi (blue).

4 CD11b GR1 CD4 CD8B220 Cell counts per field * * Myeloid cells are predominant leukocyte population found in colonic APC Min/+ tumors. Cryostat sections of colonic tumors (n=6) and small intestinal tumors (n=6) from APC Min/+ mice were stained with markers for myeloid cells (CD11b and GR), NK cells (NK1.1), T (CD4 and CD8) and B (B220) lymphocytes. Specifically stained cells were counted for 7 fields per section (40X magnification, 330μm by 270μm) using ImageJ software and then verified manually. Thereafter, cell counts were compared using Wilcoxon rank-sum test in R statistical software package. Significance level of P<0.05 is indicated by an asterisk. Supplementary figure 4

5 Small intestine SPF APC Min/+ SPF APC Min/+ IKKb(myeloid)-/- Tumor counts per mouse Colon * Tumor counts in APC Min/+ and APC Min/+ IKKb(myeloid) -/- mice Tumor load in APC Min/+ mice is reduced when IKKβ is ablated in myeloid cells. Tumor load in small intestine and colon was assessed in SPF APC Min/+ mice weeks of age (n=33) and in APC Min/+ mice with myeloid specific ablation of IKKb (n=8). Error bars represent s.d. Significance level of p<0.05 (according to Fisher exact test) is indicated by an asterisk. Supplementary figure 5 *

6 SM ST CM CT β-actin IL-1  (31 kDa pro) IL-1  (17 kDa active) Supplementary figure 6 A B C CTSTCMSM IL-1  ± ± ± ± 1.3 TNF  17.7 ± ± ± ± 1.4 IL ± ± ± ± 27.7 Expression of inflammatory cytokines in intestinal tumors of APC Min/+ mice and normal gut mucosa. (A) Western blot analysis of pro-IL-1β (31 kDa) and biologically active IL-1β (17 kDa) levels in APC Min/+ tumors from colon (“CT”) and small intestine (“ST”) and normal mucosa of colon (“CM”) and small intestine (“SM”). (B) Expression levels of IL-10 and IL-12A were analyzed in APC Min/+ tumors (CT and ST) and normal intestinal mucosa (CM and SM) by real-time PCR. (C) Relative mRNA levels of inflammatory cytokines, IL-1β, TNF-α and IL-23, in APC Min/+ tumors (CT and ST) and normal intestinal mucosa (CM and SM) are presented as mean values ± standard deviation.

7 Supplementary figure 7 CD11b+ myeloid cells in APC Min/+ colonic tumors express high levels of p-STAT3 (Tyr-705) in nucleus. Panels A-C depict (in the respective order) immunofluorescent staining of CD11b+ (green), p-STAT3 (Tyr-705) (red) and dapi (blue) in APC Min/+ colonic tumors at 40X magnification. White arrow heads indicate positively stained cells. Panel D represents merged image of all 3 colors, with cells doubly stained for CD11b and p-STAT3 appearing as orange.

8 Spleens of old APC Min/+ mice display compensatory erythropoiesis. Spleens of APC Min/+ and wild-type (APC +/+ ) mice (10 and 25 weeks of age) were fixed in 4% formalin, sectioned and stained with H&E (upper panel). An expansion of the erythroid compartment (labeled) in the spleens of APC Min/+ mice is shown. Lower panel represents analysis of anemia indicators in old APC Min/+ mice, based on four measurements from each group. Supplementary figure 8 APC Min/+ APC +/+ Hb, g/l Hematocrit Iron levels in serum APC Min/+ APC +/+ Anemia indicators in APC Min/+ mice Erythroid compartment

9 21 daysEPO PBS IP Injections FACS Tumor score Supplementary figure 9 EPO PBS CD11b+ cells from spleen BC A EPO treatment of APC Min/+ mice leads to increased myeloid (CD11b+) cell accumulation in colonic tumors and spleens. (A) A schematic representation of EPO stimulation experiments. (B) Colonic tumors from EPO or PBS treated APC Min/+ mice were snap-frozen, cryostat sectioned and immunofluorescent staining for myeloid cell marker CD11b+ (green) was performed. Cell nuclei were counterstained with dapi (blue). (C) EPO treatment results in increased recruitment of CD11b+ cells to the spleen. Amount of CD11b+ cells in spleen was measured one day following the treatment. The plot depicts proportion of CD11b+ cells in splenocyte population. T T

10 Cell counts, x10 5 Time point Supplementary figure 10

11 F4/80 CD11bIgG2b Purified Splenocyte suspention Depleted splenocyte suspention Supplementary figure 11


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