Supplementary Figure Legends

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Supplemental Figure Legends
Figure S1. Colon tumorigenesis in PG receptor-deficient mice and the effect of Ptger2
deficiency on cancer progression.
A, Representative macroscopic images of the colon in mice with each genotype subjected to
AOM/DSS treatment for 80 days. Scale bar, 8 mm. B and C, Representative HE staining of colon
tissue from Ptger1-/-, Ptger3-/- (B) or Ptges-/- (C) mice after AOM/DSS treatment for 80 days
(n=5). Scale bar, 100 m. Magnified images corresponding to the black or red box are shown in
middle or right panels, respectively. Scale bar, 20 m (upper images) or 10 m (lower images).
D, The number (left panel) and size (right panel) of colon tumors at Day 200 in wild type (n=5)
or Ptger2-/- (n=5) mice subjected to AOM/DSS treatment. * p<0.05, ** p<0.01. E, HE staining of
colon specimen collected at Day 200 from wild type (upper panels) or Ptger2-/- (lower panels)
mice subjected to AOM/DSS treatment. Magnified images corresponding to the black or red box
in left panels are shown in middle or right panels, respectively. Immunohistochemistry for SMA is also provided to visualize muscularis mucosae. White arrows or asterisks indicate the
disrupted or the intact lamina muscularis mucosae, respectively. Shown is an example of
disruption of muscularis mucosae by tumor in one out of 20 wild type mice. None of 20 Ptger2-/mice showed this invasive phenotype. Present images are otherwise representative of 20 mice in
each genotype. Scale bar, 500 m (left), 10 m (middle), 200 m (right and
immunohistorchemical image), respectively. F, Representative dot-plot of CD45+CD11b+Ly-6G+
cells (left panels) and immunostaining for Gr-1 (right panel) in the colon from wild type or
Ptger2-/- mice after AOM/DSS treatment for 80 days. Representative data from independent 3
(FACS analysis) or 5 (immunostaining) experiments is shown. Scale bar, 50 m. Note
suppression of the CD45+CD11b+Ly-6G+ cell population and infiltration of Gr-1+ cells in the
colon of Ptger2-/- mice. G, HE staining of colon specimen collected at Day 10 from wild type
(upper panels) or Ptges-/- (lower panels) mice subjected to AOM/DSS treatment. Magnified
images corresponding to the black box in left or middle panels are shown in middle or right
panels, respectively. Scale bar, 100 m (left images), 20 m (middle images) or 10 m (lower
images). H, Inflammatory cell populations in the colon of wild type or Ptger2-/- mice treated with
AOM and DSS for 50 days. The numbers of each cell types were analyzed by FACS (n=3). All
bars indicate mean±SEM. n.s. statistically not significant.
Figure S2. Specificity of anti-EP2 antibody used and the major subpopulation of Gr-1+ cells
in the colon.
A, Immunohistochemistry for EP2 (red) and nuclear staining by DAPI (blue) of colon tissue
from wild type or Ptger2-/- mice. Note the loss of EP2 signals in Ptger2-/- colon. Scale bar, 50
m. B, Immunohistochemistry for EP2 (green), Gr-1 (red) and nuclear staining by DAPI (blue)
of primary culture of neutrophils from wild type or Ptger2-/- mice. Note the loss of EP2 signals in
Ptger2-/- neutrophils. Scale bar, 5 m. C, Enrichment of Ly-6GhighLy-6Clow cells in CD11b+-cells
in the colon of wild type mice subjected to AOM/DSS treatment. Representative dot plot from 3
independent experiments is shown.
Figure S3. NF-B activation in neutrophils and EP2-dependent expression of proinflammatory genes.
A and B, Gene (A) or protein (B) expression of TNF-, IL-6, COX-2 and CXCL1 in colon from
wild type mice before (Day 0) or maintained without AOM/DSS treatment for 80 days (Day 80)
in quantitative RT-PCR analysis or immunohistochemistry, respectively. All bars indicate
mean±SEM (n=5). n.s. statistically not significant. Representative images from 5 independent
samples are shown. Scale bar, 50 m. C, Double immunohistochemistry for NF-B p65 subunit
phosphorylated at Ser468 (upper panels) or at Ser536 (lower panels) (green) and
myeloperoxidase (red) of colon specimen prepared from wild type mice after AOM/DSS
treatment for 80 days. Merged images with nuclear DAPI staining (blue) are shown in right.
Representative images from 5 independent experiments are shown. Scale bar, 50 m. D,
Representative dot plot for CD11b and Ly-6G of neutrophil preparation. Primary neutrophils
were purified with density-gradient centrifuge from bone marrow and their purity was analyzed
by FACS analysis. Representative image from 3 independent experiments is shown. E,
Expression of Ptger2 gene in primary neutrophils. mRNA was purified from primary culture of
neutrophils and the expression of Ptger2 gene was analyzed by PCR. PCR reaction without
template was served as a negative control. F, Effect of Ptger2 deficiency on TNF--or the EP2
agonist ONO-AE1-259-induced gene expressions of Il6 (left panel), Ptgs2 (middle panel) or
Cxcl1 (right panel). Neutrophils were prepared from bone marrow of wild type or Ptger2-/- mice
and stimulated with vehicle, TNF- (1 ng/ml), ONO-AE1-259 (EP2 agonist, 0.5 M) or both for
1 h. mRNA was then purified and analyzed by quantitative RT-PCR (n=3 in each group). All bars
indicate mean±SEM. *** p<0.001. Note that the effect of ONO-AE1-259 was absent in Ptger2/-
cells. G, Effect of ONO-AE1-259 on TNF--induced expressions of NF-B targeted genes in
primary culture of neutrophils. Primary culture of neutrophils was stimulated with vehicle, TNF alone (1 ng/ml) or in combination with ONO-AE1-259 (0.5 M) for 15 min (left panel), 1 h
(middle panel), or 6 h (right panel) and induction of NF-B targeted genes was analyzed in RCR
array analysis. Relative mRNA expression adjusted to one in vehicle-treated group is shown. H,
mRNA expression of Cxcl2, Cxcl3, Pf4 (Cxcl4) or Cxcl5, in the colon after AOM/DSS treatment
for 80 days and the effect of Ptger2 deficiency on these gene expressions. mRNA was purified
from the colon of wild type (n=3) or Ptger2-/- (n=5) mice before (untreated) and after AOM/DSS
treatment (AOM/DSS) for 80 days and analyzed by quantitative RT-PCR. All bars indicate mean
±SEM. * p<0.05. n.s. statistically not significant.
Figure S4. Expression of BDNF, Osteopontin and COX-2 in colon from mice without
AOM/DSS treatment.
A and B, Gene (A) or protein (B) expression of BDNF, MMP-12, osteopontin (Spp1), Wnt5a,
and COX-2 in colon from wild type mice before (Day 0) or maintained without AOM/DSS
treatment for 80 days (Day 80) in quantitative RT-PCR analysis or immunohistochemistry,
respectively. All bars indicate mean±SEM (n=5). * p<0.05. n.s. statistically not significant.
Representative images from 5 independent samples are shown. Scale bar, 50 m.

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