SB218078 Inhibits Vascular Development in Zebrafish Embryos. (A, B) Representative images of intersegmental vessels (ISVs) in zebrafish embryos at 12 h post-fertilization (hpf) treated with vehicle control and various concentrations of SB218078 (1.25 μM, 2.5 μM, 5 μM, 10 μM). (C, D) Regeneration of amputated zebrafish fins was monitored over a 7-day period. *P < 0.05, **P < 0.005, ***P < 0.001. Scale bar in (A and C) 50 μm.

SB218078 inhibits angiogenesis in vitro. (A, B) SB218078 (1 μM and 2 μM)) inhibits VEGF-induced tube formation in human umbilical vein endothelial cells (HUVECs). (C) Treatment with SB218078 (1 μM and 2 μM) for 72 h significantly reduces HUVEC proliferation. (D, F) SB218078 (1 μM and 2 μM) inhibits the formation of HUVEC colonies. (E, G–I) SB218078 also inhibits HUVEC migration and invasion. Scale bar in (A, F, and H): 50 μm.

SB218078 inhibits angiogenesis in vivo. (A) Representative images showing the sprouting of vascular endothelial cells in different groups treated with vehicle control and SB218078 (1 μM and 2 μM) in the rat arterial ring assay. (B) Quantification of the relative overall length of endothelial cell sprouting induced by SB218078 in each treatment group. (C) Representative images of the Matrigel plug assay in mice treated with vehicle control (DMSO) and SB218078 (2 μM). (D, E) Immunofluorescence staining for CD31, along with statistical analysis of microvessel density. (F, G) Kinase assays conducted to identify potential targets of SB218078 for its anti-angiogenic effects. Scale bar in (A, D): 50 μm.

SB218078 inhibits the growth of breast cancer cells in vitro. (A–C) SB218078 (1 μM and 2 μM) significantly inhibits colony formation in T47D and MDA-MB-231 cells. (D, E) Treatment with SB218078 (1 μM and 2 μM) for 72 h reduces the proliferation of T47D and MDA-MB-231 cells. (F, G) Treatment with SB218078 (1 μM and 2 μM) for 72 h promotes the cell apoptosis of T47D and MDA-MB-231 cells. (H–N) SB218078 (1 μM and 2 μM) also inhibits the migration and invasion of both T47D and MDA-MB-231 cells. Scale bar in (H, J, and L): 50 μm.

SB218078 inhibits the growth and angiogenesis of breast cancer in vivo. (A) Representative images of MDA-MB-231 cell xenografts treated with either vehicle control (DMSO) or SB218078. (B) Growth curve showing the progression of the xenografts, and (C) the corresponding weights of the xenografts. (D, E) Immunohistochemical (IHC) staining for Ki67 in xenograft tissues. (F, G) IHC analysis of microvessel density (CD31/mm²) between the two treatment groups in xenograft tissues. (H, I) Immunofluorescence assessment of microvessel density (CD31/mm²) in xenograft tissues for both groups. (J) Representative images depicting eGFP-expressing blood vessels (green) and mCherry-labeled MDA-MB-231 cells (red) following treatment of zebrafish embryos with vehicle control (DMSO) or SB218078 (2.5 μM) for 3 days. Scale bars: 200 µm. (K) Quantification of relative vascular density in each group. Scale bar in (D, F, H, and J) 50 μm.

SB218078 Inhibits the EMT in Breast Cancer Cells. (A) RNA sequencing analysis of MDA-MB-231 cells treated with SB218078. (B) Volcano plot displaying the differentially expressed genes between the vehicle control and SB218078 treatment groups. (C–F) Gene enrichment analysis indicating that SB218078 regulates various biological processes, including the cell cycle, nuclear transcription factors, and tumor-related pathways, among others.

ZEB1-Independent Effects of SB218078 on Angiogenesis. (A) SB218078 inhibits EMT-related morphological changes in MDA-MB-231 cells. (B) Immunofluorescence analysis of protein levels of Vimentin and E-cadherin in MDA-MB-231 cells treated with either vehicle control (DMSO) or SB218078 (1 μM and 2 μM). (C) Schematic representation of the designed gRNA sequences for zeb1a, snail1a, twist1a, and GFP. (D) RNA expression levels of each target in zebrafish across different treatment groups. (E–G) Knockout of ZEB1 using the CRISPR system inhibits the vascular development of intersegmental vessels (ISVs) and subintestinal vessels (SIVs) in zebrafish.