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Figure 1 3,4-Difluorobenzocurcumin treatment inhibits trunk and facial lymphatic development in zebrafish. (A?D) Lateral fluorescent images of 6 dpf Tg(-5.2lyve1b:DsRed2) larvae treated with either 0.1% DMSO (A), 20 ?M sunitinib malate (SM, B), 10 ?M curcumin (CM, C), or 2.5 ?M 3,4-Difluorobenzocurcumin (CDF, D). CDF inhibits lymphatic development in a dose-dependent manner in zebrafish. (E) Quantification of somites with thoracic duct (TD) tissue fragments in 6 dpf Tg(-5.2lyve1b:DsRed2) larvae treated with either 0.1% DMSO (n = 49 larvae), 20 ?M SM (n = 51 larvae), 10 ?M CM (n = 32 larvae), or CDF at 0.5 ?M (n = 34 larvae), 1 ?M (n = 37 larvae), 2.5 ?M (n = 48 larvae), or 5 ?M (n = 46 larvae). (F?H?) Lateral confocal images of 5 dpf Tg(fli1a:nEGFP);Tg(-5.2lyve1b:DsRed2) larvae treated with either 0.1% DMSO (F,F?), 10 ?M CRM (G,G?), or 2.5 ?M CDF (H,H?). Images (F??H?) represent the Tg(-5.2lyve1b:DsRed2) expression of images (F?H). (I,J) Quantification of TD (I) or dorsal longitudinal lymphatic vessel (DLLV, J) nuclei across 9 somites in 5 dpf Tg(fli1a:nEGFP);Tg(-5.2lyve1b:DsRed2) larvae treated with either 0.1% DMSO (n = 20 larvae), 10 ?M CM (n = 20 larvae), or 2.5 ?M CDF (n = 24 larvae). (K) Quantification of somites with intersegmental vessels (ISVs) in 48 hpf Tg(fli1a:EGFP) embryos treated with either 0.1% DMSO (n = 47 embryos), 20 ?M SM (n = 56 embryos), or CDF at 2.5 ?M (n = 35 embryos), 5 ?M (n = 39 embryos), or 10 ?M (n = 27 embryos). CDF at 2.5 ?M does not inhibit primary angiogenesis. Representative fluorescent images of graph K can be found in Figure S2A?E. (L?O?) Lateral confocal images of Tg(fli1a:nEGFP);Tg(-5.2lyve1b:DsRed2) larvae treated with either 0.1% DMSO (L,L?), 20 ?M SM (M,M?), 10 ?M CM (N,N?), or 2.5 ?M CDF (O,O?). CDF at 2.5 ?M blocks facial lymphatic development. Images (L??O?) represent the Tg(-5.2lyve1b:DsRed2) expression of images (L?O). (P?R) Quantification of lateral facial lymphatic (LFL, P), medial facial lymphatic (MFL, Q), or otolithic lymphatic vessel (OLV, R) nuclei in 5 dpf Tg(fli1a:nEGFP);Tg(-5.2lyve1b:DsRed2) larvae treated with either 0.1% DMSO (n = 14 larvae), 20 ?M SM (n = 16 larvae), 10 ?M CM (n = 14 larvae), or 2.5 ?M CDF (n = 15 larvae). Statistical test: Kruskal-Wallis test were conducted for graphs (E,I?K,P?R). PCV: posterior cardinal vein. p ? 0.001 (***) and n.s. indicates not significant. Scale bars: 100 ?m.