Figure 1

TGF-β is the acid-activated serum factor that induces branching tubulogenesis. J3B1A cells were grown in collagen gels in defined medium for 6 days to allow the formation of cystic structures. The cultures were then left untreated or were incubated with 1% acidified FCS (pH 3) for an additional 48 hours. (A) Under control conditions, J3B1A cells form spheroidal cysts enclosing a wide lumen. (B) Addition of acidified FCS induces the radial outgrowth of tube-like structures from the cyst wall. (C) Co-addition of 5 μM SB-431542, a selective inhibitor of the TGF-β type I receptor, abolishes the tubule-inducing activity of acidified FCS. (D) Pre-incubation of acid-treated FCS with a neutralizing antibody specific for TGF-β1 abrogates the tubulogenic effect of acidified FCS, whereas pre-incubation with a neutralizing antibody to TGF-β2 (E) or with a control antibody that does not react with TGF-βs (F) have no inhibitory effect. Antibodies in D-F were added at a final concentration of 5 μg/ml. Bars, 200 μm. (G) Branching tubulogenesis is suppressed by SB-431542 in a dose-dependent manner. The cultures were treated with the inhibitor two hours before addition of 1% acidified FCS. Data were expressed as mean number of outgrowths per colony ± s.e.m. from three separate experiments and statistical significance was determined using the Student's unpaired t-test. * p < 0.0025 versus values of acidified FCS alone. (H) Dose-response analysis of the effect of increasing concentrations of anti-TGF-β1 antibody. Acidified FCS (1%) was pre-incubated for 60 minutes with the indicated concentrations of the antibody before being added to the cultures. * p < 0.025 versus values of acidified FCS alone. ** p < 0.0005 versus values of acidified FCS alone.