Fig 2: Knockdown of Tspan18 by siRNA leads to downregulation of VEGFR2 and Notch1 in vitro. (A) Western blot analysis of VEGFR2 and VEGFR1 expression in Tspan18 siRNA transfected HUAEC. A mutant siRNA (Tspan18 siRNA?) was used as siRNA control. ß-actin served as loading control. (B) VEGFR2 and hNICD normalized the gene-expression changes in Tspan18-deficient HUAEC. VEGFR2 and hNICD were overexpressed in HUAEC transfected with Tspan18 siRNA. Gene expression was analyzed by quantitative RT-PCR, normalized to ß-actin level and compared to that of Tspan18 siRNA?-treated HUAEC (set as 1.0). The experiments were repeated three times with similar results. Error bars: s.d., *P<0.01, **P<0.002. P values were calculated using a two-tailed Student's t-test. (C) Western blots showing the downregulation of Notch1 and VEGFR2 in VEGFR2-expressing mesothelioma cell line 211H by Tspan18 siRNA. No change in Notch1 is observed in cervical cancer Hela cell line that has no VEGFR2. This experiment was repeated three times with similar results. The immunoblots were quantified with Image J and the relative band intensity is shown.

Fig 3: EC differentiation of hiPSCs from patients with ESRD and HCs.(A) Schematic overview of EC differentiation. (B) Expression levels of the EC markers CD31, CD34, CD133, VWF, Flk-1, and Flt in purified hiPSC-ECs before and after CD31+ isolation, as determined by immunofluorescence staining with quantitative analysis. Scale bar represents 50 µM. **p < 0.05 relative to HC-iPSC-ECs. (C) CD31+ expression on hiPSC-ECs differentiated from patients with ESRD and HCs, as determined by flow cytometry analysis. **p < 0.01 relative to HC-iPSC-ECs.EC, endothelial cell; ESRD, end-stage renal disease; Flk-1, fetal liver kinase 1; Flt-1, vascular endothelial growth factor receptor 1; HC, healthy control; hiPSC, human induced pluripotent stem cell; SSC, side scatter; vWF, von Willebrand factor.

Fig 4: Effect of VEGF-A signaling in association with the expression patterns of NRP-1, on GB cells. (A and B) Nine GB cells were incubated in the absence or presence of exogenous VEGF-A (50 µg/l, left) or SU1498 (50 µmole/l, right), a VEGF receptor inhibitor, for 72 h, after which they were examined for cell viability using WST-1 assay and cell death using LDH assay (n=3; Tukey's post hoc test was applied to significant group effects in ANOVA, P<0.0001; asterisks indicate significant differences compared with the control group, *P<0.05 and **P<0.01). (C) Efficacy of Flt-1, Flk-1, NRP-1, and VEGF-A siRNA transfections assessed by western blot analysis. GAPDH was used as a control. (D) Cell death was assessed 72 h after transient transfection with siRNAs against VEGF-A, Flt-1, Flk-1, and NRP-1 using an LDH assay (n=3; Tukey's post hoc test was applied to significant group effects in ANOVA, P<0.0001; asterisks indicate significant differences compared to 0% inhibition; *P<0.05 and **P<0.01). (E) Effect of SU1498 (10 mg/kg) on the tumor volumes of U251-MG xenograft models (control group, n=4; SU1498 10 mg/kg, n=4) measured for 42 days using the following formula: V=0.523 LW2 (L=length and W=width). Bold arrows indicate the time-points of SU1498 injection (Tukey's post-hoc test was used to determine significant group effects in ANOVA, P<0.0001; asterisks indicate significant differences between the control group and the SU1498-injected group; *P<0.05 and **P<0.01). (F) Effect of SU1498 (10 mg/kg) on the tumor volumes of LN215-MG xenograft models (control group, n=4; SU1498 10 mg/kg, n=4) measured for 42 days using the formula aforementioned, V=0.523 LW2. Bold arrows indicate the time-points of SU1498 injection. VEGF-A, vascular endothelial growth factor-A; GB, glioblastoma; NRP-1, neuropilin-1; LDH, lactate dehydrogenase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Flt-1, FMS related receptor tyrosine kinase 1; Flk-1, fetal liver kinase 1; n.s., non-significant; si-RNA or si-, small interfering RNA.

Fig 5: Tspan18 functions through VEGF/VEGFR and Notch signaling pathways. (A) Whole-mount ISH analysis of SHH, VEGFs and VEGFRs at 27 hpf in E7MO injected zebrafish. The expression of VEGFs especially VEGFaa121 and VEGFR2 was decreased (indicated by arrows) while the expression of VEGFR3 was increased in PCV (arrows) and ectopically expressed in DA (arrowheads). No significant changes were observed in SHH and VEGFR1 expression. Quantification of each genes is shown on the right (n>10). (B) Whole-mount ISH analysis of Sema3ab, PlexD1 and four Nrp isoforms in E7MO injected fish at 27 hpf. Sema3ab is highly induced in anterior somites (arrow). Quantification of each genes is shown on the right (n>10). (C) Whole-mount ISH analysis of Notch3, EphrinB2, and EphB4 at 27 hpf. In Tspan18 deficient fish, Notch3 (black arrow) and EphrinB2 (red arrow) were lost in the DA, while EphB4 was ectopically expressed in the DA (green arrow). These defects were rescued by co-injection of zebrafish VEGFR2 sense capped mRNA or Notch3 intracellular domain (N3ICD) mRNA. Quantification of each gene is shown at the bottom (n>10). (D) VEGFR2 (top) and N3ICD (middle) mRNA rescued E7MO-morphants in a dose-dependent manner (1.0, 1.5 and 2.0 ng per injection), whereas MOs targeting Sema3ab and PlexD1 (bottom) only partially rescued the defects (6 and 10 ng per injection respectively). Images of 27 hpf zebrafish are shown. Similar results were obtained from three independent experiments. All larvae lateral view. Anterior is to the left. Scale bars: 100 µm.