Gkouskou et al.: Apolipoprotein A-I inhibits experimental colitis and colitis-propelled
carcinogenesis
SUPPLEMENTARY FIGURE LEGENDS
Suppl. Figure 1: Progressive accumulation of Tyr705-phosphorylated STAT3 in the distal
colon of AOM/DSS-treated mice. Phosphorylated STAT3 immunostainings are shown for
normal epithelium (A), dysplastic epithelium at early treatment time point (B), dysplastic
epithelium at late time point (C) and tumors (D) of distal mucosa revealing the gradual increase
of p-STAT3 expression levels during carcinogenesis. In contrast, p-STAT3 remained at low
expression levels in proximal mucosa epithelial cells (lower panels). Hematoxylin was used as
counterstain; original magnification: x200.
Suppl. Figure 2: Controls for the immunohistochemical analysis of phosphorylated STAT3
expression. (A) Nuclei of malignant cells are positive for phosphorylated STAT3 (p-STAT3),
as detected by immunohistochemistry. (B) Some stroma cells in normal colon mucosa
untreated mice are p-STAT3-positive (arrow), while the nuclei of epithelial cells are negative
serving as internal positive and negative controls. Immunohistochemistry on tumor (C) and
normal colon mucosa (D) sections, after omitting primary antibody served as additional
negative controls. Original magnification x200, DAPI as chromogen, hematoxylin, as
counterstain.
Suppl. Figure 3: Progressive accumulation of p65 NF-B in the distal colon of AOM/DSStreated mice. Immunostains are shown for normal epithelium (A), dysplastic epithelium at
early treatment time point (B), dysplastic epithelium at late time point (C) and tumors (D) of
distal mucosa revealing the gradual increase of p65 expression levels (arrows) during
carcinogenesis. In contrast, p65 remained at low expression levels in proximal mucosa
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epithelial cells (lower panels). Hematoxylin was used as counterstain; original magnification:
x200.
Suppl. Figure 4: Controls for the immunohistochemical analysis of p65 NF-B expression.
(A) Spleen white pulp from the studied mice was used as positive control for activated, nuclear
p65. (B) Lymphocytes in normal colon mucosa are p65-positive (arrow), while the nuclei of
adjacent epithelial cells are negative (internal positive/negative controls). (C) Similarly, p65positive lymphocytes next to p65-positive tumor cells serve as positive internal control (arrow).
(D) Immunohistochemistry on tumor sections, after omitting primary antibody served as
additional negative control. Original magnification x400, DAB as chromogen, hematoxylin as
counterstain.
Suppl. Figure 5: Specificity of the ApoA-I antibody used for immunohistochemical analyses.
(A) Liver and proximal and distal colon tissues from WT and ApoA1-/- mice were
immunostained using an ApoA-I antibody as described in the ‘Materials & Methods’. Whereas
hepatocytes and proximal colon epithelial cells of WT mice stain positive for ApoA-I (upper
panel, dotted arrows), tissues from knock-out animals show no reactivity (middle panel).
ApoA-I, likely circulating protein, is also detected in endothelial cells (upper panel, solid
arrows). Immunohistochemistry on liver, proximal and distal colon sections from WT animals,
after omitting primary antibody serve as additional negative control (lower panel). Original
magnification: x200, DAPI as chromogen, hematoxylin, as counterstain. (B) Immunoblot
analysis of ApoA-I levels expressed in liver and proximal colon tissue from WT mice. GAPDH
serves as loading control.
Suppl. Figure 6: (A) Colon shortening, a clinical feature of intestinal inflammation in the DSS
model, is significantly more pronounced in ApoA1-/- compared to WT mice exposed to 3% DSS
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(**p<0.01; *p<0.05; Mann Whitney test). Representative photographs of the large intestine
from untreated WT and ApoA1-/- mice and animals exposed to 3% DSS for 5 days are shown in
(B).
Suppl. Figure 7: Relative levels of il1b (A) and tnf (B) mRNAs expressed in the distal and
proximal colon of WT and ApoA-I-deficient mice exposed to DSS versus untreated control
animals, normalized to actin. ***p<0.001; **p<0.01; Mann Whitney test.
Suppl. Figure 8: Circulating levels of ApoA-I (A) and HDL (B) in ApoA1+/+ (n=4), ApoA1+/(n=5) and ApoA1-/- (n=5) mice. **p<0.01; *p<0.05; Mann Whitney test.
Suppl. Figure 9: Relative levels of il6 mRNA expressed in the distal colon of WT and
ApoA1+/- mice exposed to 3%DSS for 5 days versus untreated control animals, normalized to
actin. **p<0.01; Mann Whitney test.
Suppl. Figure 10: Representative immunohistochemical analysis of p65 NF-B in colon
tumors developed in AOM/DSS-treated WT and ApoA1+/- mice.
Supplementary Table Legends
Suppl. Table 1: Biological processes (Gene Ontology; GO) annotation related to gene
expression of proximal (A) and distal (B) colon (p<E-03).
Suppl. Table 2: Biological processes (Gene Ontology; GO) annotation related to CAC gene
signature.
Suppl. Table 3: KEGG pathways related to the CAC gene signature. The CAC genes identified
to be associated with each KEGG are shown in column G.
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Suppl. Table 4: Biological processes (Gene Ontology; GO) annotation related to resistanceassociated gene signature.
Suppl. Table 5: KEGG pathways related to resistance-associated gene signature. The signature
genes identified to be associated with each KEGG are shown in column G.
Suppl. Table 6: Predicted activation state of transcription factors (upstream regulators) in the
distal colon of late (A) and early (B) stages of CAC. Light brown color denotes predicted
activation and blue color denotes predicted inhibition of transcription factor activity.
Suppl. Table 7: Predicted activation state of transcription factors associating with resistance to
AOM/DSS in the proximal colon.
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