Figure 5

Dysplastic epithelium in gastric organoids. (a) Gastric organoid cultures were made from gastric tissue of neonatal mice harboring Cdh1 and Trp53 floxed alleles then subsequently infected with Fc-expressing adenovirus, or CreGFP-expressing adenovirus +/- retrovirus expressing shRNA against Tgfbr2. Images indicate immunofluorescence with nuclear DAPI staining and antibodies against CDH1, TGFBR2, or PCNA. Intrinsic GFP fluorescence from adenovirus CreGFP was abrogated by tissue passaging and subsequent formaldehyde fixation, and is not visible in immunofixation experiments. (b) GSM-06 murine gastric epithelial cells were infected with scrambled shRNA or an shRNA against murine Tgfbr2 and lysates probed by western blotting with antibodies against TGFBR2 or β-actin. (c) Gastric organoid cultures were made from gastric tissue of neonatal mice harboring Cdh1 and Trp53 floxed alleles and subsequently infected with retrovirus expressing shRNA against Tgfbr2. Images are of cultured spheres at days 2 and 20. (d) Images represent H&E stained gastric organoids with the indicated genotypes taken at low (40×) or high power (400×).