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Fig. 3. Sema3A and Sema3C additively promote SMG cleft formation. (A) Sema3A and Sema3C are the only class 3 semaphorins that could promote cleft formation in the SMG co-culture assays. Representative photographs of the growth of SMG explants co-cultured with class 3 semaphorins were shown. The bar graph summarizes the ratios of the number of terminal buds in each co-culture to the number of the terminal buds in the control culture (n7). The number of terminal buds at the presence of Sema3A or Sema3C was almost doubled. (B) Treatment of antisense ODNs against Sema3A or Sema3C specifically reduced the number of terminal buds in the SMG cultured ex vivo. Representative explants are shown. The bar graph summarizes five independent experiments. The terminal bud number was significantly further reduced when both Sema3A antisense ODNs and Sema3C antisense ODNs were added together into the culture. Paired t-test: ^*, P<0.05; ^**, P<0.01. (C) Sema3A and Sema3C additively promoted bud formation in a concentration-dependent manner. In the SMG co-culture experiments, Sema3-transfected COS cells were serially diluted to test the synergistic effects. Fold dilution in the co-culture is indicated as the Sema3-transfected COS cells diluted with mock-transfected COS cells. (a) Additive effects of Sema3A and Sema3C were most obvious at the lower concentrations of semaphorins. The effects were saturated at 1:1 dilution. (b) The amounts of semaphorin proteins present in each condition were assayed by western blotting. Gradual decreases of Sema3A or Sema3C in the serial dilutions were observed. Tubulin: internal loading control. (c) The bar graph summarizes four independent experiments. Scale bars: 100 µm. C, scrambled sequence.

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