Fig 1.
(A) Schematic representation of LPA2 TG construct and genotyping of TG mice using two sets of primers are shown. (B) The entire intestinal tract of LPA2 TG and WT mice are shown. Bar: 1 cm.
Fig 2.
TG expression of LPA2 in the intestinal tract results in dysplasia.
H&E staining of small intestine (jejunum and ileum) and colon (proximal and distal) of LPA2 TG and WT mice are shown. Magnification: x100. Right panels show high power images. Bar: 100 μm. n = 3 per group.
Fig 3.
Expression of VSVG-LPA2 in mice.
(A) TG VSVG-LPA2 expression in small intestine (SI, left) and colon (right) of LPA2 TG (upper two panels) and WT (lower panel) mice was determined. Anti-VSVG antibody was used for immunohistochemistry. Magnification: x100. Lower panels show magnified views (x400) of the boxed area. WT was used as a negative control. Bar: 100 μm. (B) VSVG-LPA2 expression in the small intestine and colon of TG mice was assessed by immunofluorescence microscopy. Bar: 100 μm. Images were captured under identical setting to compare the relative expression levels of VSVG-LPA2 in small intestine and colon. Integrated fluorescence signal intensity was averaged over the area of epithelial cell layer. Lower graph shows the relative fluorescence signal intensity. *, p < 0.01, small intestine versus colon. n = 3 per group. Statistical analysis was performed using 2-tailed Student’s t test.
Fig 4.
LPA2 overexpression results in opposite effects in the small intestine and colon.
IEC proliferation (A) and apoptosis (B) were determined by immunohistochemical analysis for Ki67 and cleaved caspase-3, respectively. Representative images of TG and WT mice are shown Magnification: x200. Arrows point to apoptotic cells positive for cleaved caspase-3. Magnification: x200. Bar: 100 μm. Right, Ki67 proliferation and cleaved caspase-3 index are shown. The number of Ki67+ cells per crypt was quantified from thirty crypts per mouse. The level of apoptosis was expressed as percentage of apoptotic cells. Twenty fields of vison per mouse were counted. * P < 0.01. n = 3. Statistical analysis was performed using 2-tailed Student’s t test.
Fig 5.
LPA2 overexpression does not affect β-catenin expression.
β-catenin expression in the small intestine (SI) and colon was compared between TG and WT mice. Representative images of β-catenin staining are shown.Bar: 100 μm. n = 3.
Fig 6.
LPA2 overexpression results in opposite effects of KLF5 expression in the small intestine and colon.
KLF5 expression in the small intestine (SI) and colon of TG and WT mice are shown. Magnification: x200. Bar: 100 μm. n = 3.
Fig 7.
Absorptive epithelial cells in LPA2 TG intestinal tract are poorly differentiated.
(A) IAP staining in the small intestine was determined by a chromogenic assay. Red color indicates IAP activity. Magnification: x200. Bar: 100 μm. (B) Immunohistochemically staining of NHE3 on the surface membrane of the small intestine and colon is shown. In comparison to WT, NHE3 staining in TG intestine was discontinuous and less uniform. Arrows mark NHE3 label on the membrane. Magnification: x200. Bar: 100 μm.
Fig 8.
Decreased number of Paneth cells and enteroendocrine cells in LPA2 TG intestinal tract.
Goblet (A), Paneth (B), and enteroendocrine (C) cells were identified by Alcian blue, lysozyme, and Chromogranin A expression, respectively. Magnification: x200. Bar: 100μm. Arrow, enteroendocrine cells. Goblet, Paneth, and enteroendocrine cells were quantified by counting alcian blue-positive, lysozyme-positive, and ChrA-positive cells per villus or crypt, respectively. Fifty villi or crypts were quantified for each mouse. The graphs show the number of each cell type (mean ± SEM). Statistical analysis was performed using 2-tailed Student’s t test. * P < 0.05, ** P < 0.01.
Fig 9.
LPA2 overexpression correlates with the occurrence of dysplasia in founder mice.
TG VSVG-LPA2 expression was determined in founder mouse (F0) small intestine and colon. Left panels show representative IHC staining using anti-VSVG antibody. Right panels show H&E of serial sections. Magnification: x200. Insets show a magnified view (x400) of the boxed area. Bar: 100 μm. n = 4.