Mutant Lgr5+ progenies rapidly expanded and failed to differentiate. (A) Double IF analysis using RFP (for recombined cells) and GFP (for Lgr5+ cells) antibodies in control as well as Smad4 and PTEN double-mutant glands 30 days after induction. Quantification of the Lgr5+ cell number per RFP+ gland is shown below. (B) Double IF analysis using RFP and Ki67 antibodies in control and double-mutant glands 30 days after induction. Quantification of the Ki67+ cell number per RFP+ gland is shown below. (C-E) Double IF analysis of RFP and differentiated cell markers, such as Pepsinogen (C, for chief cell), DBA (D, for parietal cell) and UEA (E, for pit cell) in control and double-mutant glands. (F) Double staining of RFP by IF and Gastrin (for hormone-secreting G cell) mRNA by in situ hybridization in control and double-mutant glands. Significance was calculated using Student's t-test (n = 4). Scale bar, 50 μm.