(a) Reverse sequence analysis for exon 3 of TGFBR2 in the cell lines Lovo (Upper) and Caco2 (Lower). Lovo shows both a single- and double-base pair deletion in the A10 tract (arrow). Caco2 shows the corresponding wild-type sequence. (b) Histogram showing the changes in absorbance reading in response to TGF-β stimulation in the four cell lines (Lovo, SW48, Colo205, and Hca46) that were responsive (Left) and four cell lines (Colo320, HCA7, DLD1, andCaco2) which were unresponsive (Right). Each experiment was performed in quadruplicate, and means thus derived were used for comparison. Two of the responsive cell lines (Lovo and SW48) were RER+ with TGFBR2 mutations and two (Colo205 and HCA46) were RER−, without evidence of TGFBR2 mutations. All four responsive cell lines showed a significant reduction in cell mass when cultured in the presence of TGF-β (two-tailed t test): Lovo, P = 0.0007; SW48, P = 0.02; Colo205, P = 0.002; HCA46, P = 0.01. Error bars indicate standard deviation. (c) Growth curve for Lovo over a 36-hour period, plotted at 0, 12, 24, and 36-hour time points. Cells were grown in the absence of TGFβ1 (a); in the presence of TGF-β1 and anti-TGFβ1 antibody (b); or in the presence of TGF-β1 only (c). Growth inhibition in response to TGF-β1 was seen in Lovo after 12 hours (P = 0.009). The addition of anti-TGF-β1 antibody to the culture negated the growth-inhibitory effects of TGF-β1, thus demonstrating the specificity of the response. Error bars indicate standard deviation. (d) SSCP analysis of exon 3 in TGFBR2 for a human random control (HRC), SW48, Lovo, and DLD-1. This exon has a poly(A) tract consisting of A10 (wild-type) in HRC, A9 in SW48, A8 and A9 in Lovo, and A9 and A10 in DLD1. Each allele showed specific band mobility, and double bands were seen in those cell lines containing two different alleles. All four samples had unique banding patterns, and there was no trace of an A10 (wild-type) allele in SW48 and Lovo (the two responsive RER+ cell lines). (e) Scatterplot of proportional changes in E-cadherin and γ-catenin in response to TGF-β1 stimulation in growth medium supplemented with 10% FCS. The correlation coefficient was 0.68.