Figure 1.
Histological analysis of normal intestine in FHL2−/− mice and intestinal adenomas in ApcΔ14/+FHL2−/− mice.
A. Normal intestinal architecture in FHL2−/− mice. Intestine and colon sections from 11 month-old wt and FHL2−/− mice were stained with H&E or Ki-67 by immunostaining. B. Intestinal adenomas in ApcΔ14/+FHL2+/+ and ApcΔ14/+FHL2−/− mice. Original magnifications, X100 (A and B).
Figure 2.
Loss of FHL2 reduces intestinal polyp multiplicity.
(A) Representative images of intestines from 11-month-old ApcΔ14/+FHL2+/+ and ApcΔ14/+FHL2−/− mouse. Note the marked decrease in the number of polyps in ApcΔ14/+FHL2−/− mice. (B) Total number of intestinal polyps was counted in 11 ApcΔ14/+FHL2+/+, 16 ApcΔ14/+FHL2−/+ and 18 ApcΔ14/+FHL2−/− mice at 11-month-old. Compared to ApcΔ14/+FHL2+/+ littermates, significant reduction in polyp number was observed in ApcΔ14/+FHL2−/+ (p<0.0046) and ApcΔ14/+FHL2−/− mice (p<0.0001). (C) Polyp distributions are expressed as the percentages of mice having 0–9, 10–19, 20–29, 30–39 or more than 40 polyps in the intestine. (D) FHL2 deficiency had no effect on tumor growth. The percentages of polyps in each mouse with sizes less than 2 mm, between 2 to 5 mm and more than 5 mm are presented.
Figure 3.
Activation of the Wnt signalling in the intestine of ApcΔ14/+FHL2−/−mice.
(A) Immunostaining for β-catenin in adenomas (a, b), for cyclin D1 in adenomas (c, d) and normal intestine (e, f) and for c-myc in normal intestine (g, h) in ApcΔ14/+FHL2+/+ (a, c, e, g) and ApcΔ14/+FHL2−/− mice (b, d, f, h). Sections were photographed at the same magnification. Original magnifications: X200. (B) Activation of the Wnt target genes cyclin D1 and c-myc in adenomas is unaffected by FHL2 deletion. Real-time RT-PCR analysis of cyclin D1 and c-myc expression was performed with intestinal tumor (T) and adjacent normal tissue (NT). Cyclin D1 and c-myc expression was normalized to 18S RNA and normalized values in normal intestine of ApcΔ14/+FHL2+/+ was arbitrarily set at 1. The average values and standard deviations from two independent experiments from four mice of each genotype are shown. The p value is calculated according to Student's test.
Figure 4.
FHL2 deficiency accelerates enterocyte movement.
(A) Representative images of BrdU-positive cells along the crypt-villus axis at 2 h and 48 h post-BrdU labelling in ApcΔ14/+FHL2+/+ and ApcΔ14/+FHL2−/− mice. (B) Distribution of BrdU-positive cells at 48 h. The crypt base was set as position 0. Three mice of each genotype were analyzed. (C) Representative images of BrdU-positive cells along the crypt-villus axis at 48 h post-BrdU labelling in wt and FHL2−/− mice. Original magnifications: X200 (A and C).
Figure 5.
Up-regulation of FHL2 in intestinal tumors from ApcΔ14/+ mice and human colon tumors.
(A) FHL2 immunostaining of normal intestine and adenoma from ApcΔ14/+ mouse. Original magnifications, X200. (B) Quantitative RT-PCR analysis of FHL2 expression in adenoma and adjacent normal tissue from five ApcΔ14/+ mice. FHL2 expression was normalized to 18S RNA. The ratio of the FHL2/18S signal in NT was arbitrarily set at 1. The average values and standard deviations for three independent experiments from five ApcΔ14/+ mice are shown. Average fold induction in adenomas is 1.73 (p = 0.011, Student's t test). (C) FHL2 expression in normal human colon (a), in human low-grade (b) and high-grade colon dysplasia (c) and in human colon carcinoma (d) by immunohistochemistry. Control staining on high-grade dysplasia and human colon carcinoma was performed as FHL2 staining without the primary anti-FHL2 antibody. Original magnifications, X200. Part of the image of human colon carcinoma was further enlarged to better visualize individual cells (e).