Fig 2: ALK1 inhibitor ameliorates LPS-induced mortality and experimental metastasis of melanoma cells in mice. (A) Lung colony number in the experimental metastasis models using wild-type mice treated with or without K02288. (n = 4, *P < 0.05 by the unpaired t test). (B) Lung colony number in the experimental metastasis models using Robo4−/− mice treated with or without K02288. (n = 4, N.S., not significant by the unpaired t test). (C) Survival study of LPS-injected wild-type (C57BL/6N) mice treated with or without K02288 (control: n = 17, K02288: n = 18; P = 0.012 by the log-rank test). (D) Survival study using LPS-injected Robo4−/− mice treated with or without K02288 (n = 10; P = 0.523 by the log-rank test). Data are expressed as the mean ± SEM.

Fig 3: ALK1 inhibitor increased Robo4 expression and suppresses LPS-induced vascular permeability in mice. (A) ALK1 expression levels in organs of C57BL/6 mice (n = 4). (B) Robo4 expression levels in lungs and kidneys of wild-type (C57BL/6N) mice treated with or without K02288 (n = 7, *P < 0.05, N.S., not significant by the unpaired t test). (C) Vascular permeability assay using wild-type mice treated with or without K02288. Extravasated Evans blue in lungs and kidneys was quantified. (n = 8, *P < 0.05; N.S., not significant by the unpaired t test). (D) Vascular permeability assay using Robo4−/− mice treated with or without K02288. Extravasated Evans blue in lungs and kidneys was quantified. (n = 5, N.S., not significant by the unpaired t test). Pictures are representative images of the lungs and kidneys. Data are expressed as the mean ± SEM.

Fig 4: Competitive SMAD signaling pathways regulate Robo4 expression both positively and negatively. (A) Robo4 expression levels in HUVECs treated with siRNA for SMAD2 and SMAD3 (n = 4, **P < 0.01 by the unpaired t test). (B) Robo4 expression level in TGF-β1/2-treated HUVECs under normal (n = 4) or Matrigel conditions (n = 5, *P < 0.05, **P < 0.01 by Dunnett’s test). (C) Robo4 expression levels in HUVECs treated with BMP9 (n = 4, **P < 0.01 by the unpaired t test). (D) Robo4 expression levels in HUVECs treated with BMP9 and siRNA for SMAD1 and SMAD5 (n = 4, **P < 0.01; N.S., not significant by Tukey’s test). (E) Robo4 expression levels in HUVECs treated with K02288 and BMP9 (n = 4, **P < 0.01 by Tukey’s test). (F) Robo4 expression levels in HUVECs cultured in Matrigel containing K02288 (n = 4, **P < 0.01; N.S., not significant by the unpaired t test). (G) Schematic illustration of positive and negative Robo4 regulation by TGF-β-ALK5-SMAD2/3 and BMP9-ALK1-SMAD1/5 signaling pathways. Data are expressed as the mean ± SEM.