Fig. 7.

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 siRNAΔ) was used as siRNA control. β-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 β-actin level and compared to that of Tspan18 siRNAΔ-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.