Fig. 6. Effect of HGF and other factors on tubule formation. (A) Comparison of the frequency of tubule formation in response to HGF and EGF. Matrigel cultures were initiated in Phenol Red-free medium supplemented with 5 µg/ml insulin, 5 µg/ml transferrin, 5x10^–8 M hydrocortisone, and either 40 ng/ml HGF, 10 ng/ml EGF, 1 ng/ml TGF-ß, EGF + TGF-ß, or no further supplement. The frequency of tubule formation was determined as described in Materials and Methods. The tubules that formed in response to HGF were observed (B) under the inverted microscope, as well as (C) under the confocal microscope after incubation with Lucifer Yellow and DiI as described in the legend to Fig. 3, with the exception that HGF, rather than EGF, was utilized. Bars, 50 µm (B,C). The effect of 5x10^–5 M PD98059 (PD) and 5x10^–8 M wortmannin (Wort) on the frequency of tubule formation was examined either (D) in the presence or in the absence of 10 ng/ml EGF, or (E) in the presence or in the absence of 40 ng/ml HGF. Results were significantly (P<0.05) different from the control (^*), EGF (^**) or HGF (^**).