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Fig. 6
The functional connection between dynamin-dependent endocytosis and fscn1a during endoderm formation.
(a) The inhibitory effect of dynasore on the endocytosis of membrane Myc-ALK5. NIH3T3 cells transfected with Myc-tagged ALK5 (1 µg in one well of six-well plate) were incubated with or without 80 µM dynasore for 30 min before antibody-labelled TGF-β type I receptor endocytosis assays. Then cells were incubated with anti-Myc antibody at 4 °C for 5 h, followed by incubation with PBS containing 10% knockout serum replacement (KSR) at 37 °C for 30 min. Dynasore were added during these incubation processes. The cells were visualized by immunofluorescence with anti-Myc (green) antibody. Scale bar, 5 µm. (b–d) The expression of sox32 and gata5 were assessed by in situ hybridization (b) and real-time PCR (c) at the shield stage in zebrafish embryos treated with 80 µM dynasore or injected with 400 pg of dynamin K44A mRNA. The expression of mesodermal (ntl, gsc and eve1), non-neural ectodermal (gata2) and neuroectodermal (otx2) markers were also examined by real-time PCR in these embryos (d). In c and d, the data are presented as mean±s.d. of three independent experiments. Student’s t-test, *P<0.05. NS, non-significant. The expression of β-actin was used as a reference to normalize the amount of mRNAs in each sample. (e) NIH3T3 cells transfected with the indicated plasmids were treated with or without TGF-β1 (5 ng ml-1) and dynasore (80 µM) for 12 h, and then collected for luciferase measurements. The relative luciferase activity was the mean with SD from three independent experiments. Student’s t-test, *P<0.05. (f) Inhibition of dynamin-dependent endocytosis impairs the fscn1a mRNA injection-induced rescue effects in fscn1a morphants. Embryos were co-injected with fscn1a MO1 and the indicated mRNAs at the one-cell stage and harvested at the shield stage for sox32 detection.