Canthin-6-one inhibits intersegmental vessel and sub-intestinal vessel development in zebrafish. (A–J) Lateral fluorescent images of 48 hpf (A–E) and 4 dpf (F–J) Tg(fli1a:EGFP) embryos treated with either 0.2% DMSO (A,F), 20 µM sunitinib malate (SM, B,G), or canthin-6-one at 10 µM (C,H), 15 µM (D,I), or 20 µM (E,J). White arrows indicate the intersegmental vessel (ISV, A) or the sub-intestinal vessel (SIV, F). Red asterisks indicate the vascular compartments within the SIV (F,H,I). White asterisks indicate the reduced/absence of ISVs (B,D,E) or SIV (G,I,J). (K) Quantification of the number of fully formed ISVs in 48 hpf Tg(fli1a:EGFP) embryos treated with either 0.2% DMSO (n = 30 embryos), 20 µM SM (n = 29 embryos), or canthin-6-one at 10 µM (n = 20 embryos), 15 µM (n = 29 embryos), or 20 µM (n = 37 embryos). (L) Quantification of the length of ISVs in 48 hpf Tg(fli1a:EGFP) embryos treated with either 0.2% DMSO (n = 32 embryos), 20 µM SM (n = 26 embryos), or canthin-6-one at 10 µM (n = 25 embryos), 15 µM (n = 20 embryos), or 20 µM (n = 37 embryos). (M) Quantification of the number of SIV compartments in 4 dpf Tg(fli1a:EGFP) larvae treated with either 0.2% DMSO (n = 23 larvae), 20 µM SM (n = 20 larvae), or canthin-6-one at 10 µM (n = 22 embryos), 15 µM (n = 21 embryos), or 20 µM (n = 24 embryos). Statistical test: Kruskal-Wallis test was conducted for graphs (K–M). p ≤ 0.001 (***), p ≤ 0.01 (**), p > 0.05 (not significant, n.s.). Scale bars: 100 µm.

Canthin-6-one inhibits endothelial cell proliferation in zebrafish and HUVECs. (A,B’) Lateral confocal images of 48 hpf Tg(fli1a:EGFP);Tg(fli1a:H2B-mCherry) embryos treated with either 0.2% DMSO (A,A’) or 20 µM canthin-6-one (B,B’). Images (A’,B’) represent the fli1a:H2B-mCherry expression of images (A,B). (C) Quantification of intersegmental vessel (ISV) endothelial cell number in 48 hpf Tg(fli1a:EGFP);Tg(fli1a:H2B-mCherry) embryos treated with either 0.2% DMSO (n = 22 embryos) or 20 µM canthin-6-one (n = 21 embryos). Each data points represent an average of 5 ISVs from one embryo. (D) Quantification of relative percentage of proliferating HUVECs upon treatment with either 0.2% DMSO (n = 48 views), 10 µM sunitnib malate (SM, n = 30 views), or canthin-6-one at 10 µM (n = 28 views), 20 µM (n = 46 views), or 40 µM (n = 30 views). Results from at least 3 independent replicates. (E) Representative images of HUVECs treated with either 0.2% DMSO, 10 µM SM, or canthin-6-one at indicated concentrations. Cells were stained with Hoechst 33,342 (blue) and Alexa Fluor 488 (red) using the Click-iT assay. Blue indicates viable cells and red indicates proliferating cells. Statistical test: Mann-Whitney test for (C) and Kruskal-Wallis test for (D). p ≤ 0.0001 (****), p ≤ 0.001 (***), p > 0.05 (not significant, n.s.). Scale bars: 100 µm (A), 250 µm (E).

Canthin-6-one does not inhibit the VEGFA/VEGFR2 pathway. (A–C’’) Lateral spinning disc confocal images of intersegmental vessel (ISV) endothelial cells in 28 hpf Tg(fli1aep:ERK-KTR-Clover);Tg(fli1a:H2B-mCherry) embryos treated with either 0.2% DMSO (A,A’,A’’), 20 µM SM (B,B’,B’’), or 20 µM canthin-6-one (C,C’,C’’). Images (A–C) show fli1aep:ERK-KTR-Clover expression, while images (A’–C’) show both fli1aep:ERK-KTR-Clover and fli1a:H2B-mCherry expression. Images (A’’–C’’) show the nuclear fli1aep:ERK-KTR-Clover expression with intensity differences represented in 16 colour LUT (Fiji). The fli1a:H2B-mCherry signal was used to mark the nucleus. (D) Quantification of nucleus/cytoplasm ERK-KTR-Clover intensity in leading ISV endothelial cells of 28 hpf embryos treated with either 0.2% DMSO (n = 48 ISV endothelial cells from 10 embryos), 20 µM SM (n = 42 ISV endothelial cells from 10 embryos), or 20 µM canthin-6-one (n = 45 ISV endothelial cells from 12 embryos). (E) Western blot analysis of lysates isolated from HUVECs treated with either 0.2% DMSO, 10 µM sunitinib malate (SM), or canthin-6-one at indicated concentrations for 1 h and stimulated with vascular endothelial growth factor A (VEGFA) for 10 min (n = 3). Protein levels of pVEGFR2, total VEGFR2, or Tubulin were assessed. The full-length blots are presented in Figure S6. Statistical test: Kruskal-Wallis test was conducted for graph (E). p ≤ 0.0001 (****), p > 0.05 (not significant, n.s.). Scale bar: 50 µm (A).

Canthin-6-one inhibits tumour-associated angiogenesis in zebrafish. (A–C) Lateral light-sheet microscope images of 2 dpt (4 dpf) Tg(fli1a:EGFP) larvae injected with B16F10 melanoma cells and treated with either 0.2% DMSO (A), 20 µM sunitinib malate (SM, B), or 20 µM canthin-6-one (C) from 2 dpf. White arrow heads show tumour-associated blood vessels and asterisks indicate the absence of tumour-associated angiogenesis. (D,E) Quantification of blood vessels within the B16F10 tumour mass relative to tumour mass volume (D) or tumour mass volume (E) in 2 dpt (4 dpf) Tg(fli1a:EGFP) larvae injected with B16F10 melanoma cells and treated with either 0.2% DMSO (n = 38 larvae), 20 µM SM (n = 23 larvae), or 20 µM canthin-6-one (n = 30 larvae). Statistical test: Ordinary one-way ANOVA test was conducted for graphs (D,E). p ≤ 0.0001 (****), p ≤ 0.05 (*), p > 0.05 (not significant, n.s.). Scale bar: 100 µm (A).

Canthin-6-one synergises with VEGFR inhibitor sunitinib malate. (A–E’) Lateral fluorescent (A–E) and trans-light images (A–E’) of 48 hpf Tg(fli1a:EGFP) embryos treated with either 1% DMSO (A,A’), 20 µM sunitinib malate (SM, B,B’), 5 µM SM (C,C’), 10 µM canthin-6-one (D,D’), or co-treatment of 5 µM SM and 10 µM canthin-6-one (E,E’). White asterisks indicate the absence of intersegmental vessels (ISVs). (F,G) Quantification of the percentage of fully formed ISVs (F) or length of ISVs (G) in 48 hpf Tg(fli1a:EGFP) embryos treated with either 1% DMSO (n = 23), 20 µM SM (n = 22), 5 µM SM (n = 23), 10 µM canthin-6-one (n = 17), or co-treatment of 5 µM SM and 10 µM canthin-6-one (n = 21). Statistical test: Kruskal-Wallis test was conducted for graphs (F,G). p ≤ 0.0001 (****), p ≤ 0.001 (***), p ≤ 0.01 (**), p ≤ 0.05 (*), p > 0.05 (not significant, n.s.). Scale bar: 1 mm (A).