Figure 1

Generation of Inha/Smad2 cKO mice. (A) Smad2^flox/+; Inha^+/-mice were mated with Smad2^flox/floxmice to produce Smad2^flox/flox; Inha^+/-mice, and Smad2^+/-mice were mated with Inha^+/-; Amhr2^cre/+mice to generate Smad2^+/-; Inha^+/-; Amhr2^cre/+mice. These mice were then crossed to produce Smad2^flox/-; Inha^-/-; Amhr2^cre/+mice (experimental group) and Smad2^flox/-; Inha^-/-mice (control group). The generation of the Inha null, Smad2 null and Smad2 floxed alleles is described in the Materials and Methods. (B) Genotyping of mice using PCR analysis. Representative PCR images are shown with corresponding genotypes listed below: 1. Inhα^-/-; Smad2^flox/-; Amhr2^+/+; 2. Inhα^-/-; Smad2^flox/-; Amhr2^cre/+ ; 3. Inhα^+/+; Smad2^flox/+; Amhr2^cre/+; 4. Inhα^+/-; Smad2^flox/+; Amhr2^cre/+; 5. Inhα^+/-; Smad2^flox/+; Amhr2^+/+. (C) Recombination of Smad2 floxed allele in Inhα^-/-; Smad2^flox/-; Amhr2^cre/+tumors. Note that the recombined (Rec) allele of Smad2 can be detected only in the tumor tissues of the Inha/Smad2 cKO mice but not in the controls where the Cre recombinase is not expressed. (D and E) Immunohistochemical analysis of SMAD2 protein expression in Smad2^flox/-; Inha^-/-; Amhr2^+/+(D) and Smad2^flox/-; Inha^-/-; Amhr2^Cre/+(E) mice. Both panels are representative images. Note the dramatic reduction of SMAD2 protein expression in the conditional knockout mice vs. controls (100 × magnification).