Fig. 4

Chemical screening with Tg(snai1b:GFP) identifies TP-0903 as an inhibitor of NC EMT. (A) Dorsal views of Tg(snai1b:GFP) embryos treated with DMSO (top) or TP-0903 (bottom) at 11.5hpf and imaged at the indicated time points by 10× confocal time-lapse microscopy. TP-0903-treated embryos accumulate GFP-positive cells in the dorsal neural tube (arrowhead) and lateral cells remain associated with the neural plate border and are fused (19/20 embryos) (compare numbered streams in DMSO control to numbered asterisks in TP-0903). E, eye. (B) Transverse sections of DMSO (top)- and TP-0903-treated (bottom) Tg(snai1b:GFP); Tg(sox10:RFP) embryos at 24hpf, showing that TP-0903 causes accumulation of GFP-positive cells in the dorsal neural tube (4/5 embryos). (C) Transverse sections through hindbrain of Tg(snai1b:GFP) embryos at 24hpf treated with DMSO (top) or TP-0903 (bottom) and analyzed by immunofluorescence for pan-Cadherin and GFP, showing that GFP-positive cells in TP-0903-treated embryos maintain Cadherin levels and cell-cell adhesion within the neural tube (3/4 embryos). (D) Dorsal views of DMSO (left)- and TP-0903-treated (right) embryos at 18hpf processed for twist1a mRNA in situ hybridization, showing that TP-0903 significantly reduces twist1a expression levels (18/20 embryos) (compare numbered streams in DMSO to numbered asterisks in TP-0903). (E) Analysis of TWIST1 and SNAIL1 mRNA expression in DMSO- and TP-0903-treated PANC-1 cells by RT-qPCR. Two-way ANOVA was used to measure statistical significance. (F) Western blot analysis of PANC-1 cells treated with DMSO or 0.5-10µM TP-0903, showing a dose-dependent decrease in SNAIL1 protein expression.