Figure 3

3,4-Difluorobenzocurcumin treatment attenuates VEGFC-induced phosphorylation of ERK in endothelial cells. (A) Western blot analysis of lysates isolated from human dermal lymphatic microvascular endothelial cells (HMVECs) treated with either 0.05% DMSO, 5 ?M sunitinib malate (SM), 3,4-Difluorobenzocurcumin (CDF) at indicated concentrations, or 5 ?M curcumin (CM) for 2 h and stimulated with vascular endothelial growth factor C (VEGFC) for 20 min (n ? 4). Protein levels of pERK1/2, total ERK1/2, pAKT, total AKT, and Tubulin were assessed. CDF treatment results in a dose-dependent reduction of phosphorylated ERK (pERK) level. The full-length blots are presented in Figure S4. (B?C?) Lateral confocal images of 32 hpf Tg(fli1a:nEGFP) embryos treated with either 0.1% DMSO (B) or 2.5 ?M CDF (C) immunostained with anti-pErk (red) and anti-GFP (green) antibodies. CDF blocks phosphorylation of Erk in venous endothelial cells in vivo. Images (B?,C); represent the anti-pErk staining of images (B,C). (D) Quantification of pErk and fli1a:EGFP-positive nuclei per somite in the posterior cardinal vein (PCV) of 32 hpf Tg(fli1a:nEGFP) embryos treated with either 0.1% DMSO (n = 19 embryos) or 2.5 ?M CDF (n = 21 embryos). (E) Western blot analysis of lysates isolated from 3 dpf zebrafish larvae treated with either 0.1% DMSO, 20 ?M SM, 2 ?M PD0325901, CDF at indicated concentrations, or 10 ?M CM (n = 4). CDF is not a general inhibitor of Erk phosphorylation. Protein levels of pErk1/2, total Erk1/2, and Actin were assessed. The full-length blots are presented in Figure S5. Statistical test: Mann-Whitney test was conducted for graph (D). p ? 0.001 (***). Scale bar: 50 ?m.