Fig. 4. MAM-mediated angiogenesis is dependent on VEGFR2.
a Angiogenesis PCR array for FUNDC1f/YCdh5− and FUNDC1f/YCdh5+ endothelial cells. The heat map depicts relative expression values for the 30 discrepant genes (n = 3 biologically independent samples per group). b HUVECs were transfected with Scr siRNA or FUNDC1 siRNA for 24 h, the protein level of vascular endothelial growth factor receptor 2 (VEGFR2) were determined by western blot assays (n = 5 independent experiments). c HUVECs were infected with Ad-null or Ad-FUNDC1 for 24 h, and protein level of VEGFR2 were determined by RT-qPCR and western blot assays (n = 5 independent experiments). d HUVECs were transfected with Scr siRNA or FUNDC1 siRNA for 24 h and then infected with adenovirus encoding control (Ad-Cont) or mitochondrial–endoplasmic reticulum (ER) linker (Ad-Linker) for another 24 h (n = 5 biologically independent samples per group). Expression of VEGFR2 and FUNDC1 was determined by western blot assay. e HUVECs were transfected with Scr siRNA or FUNDC1 siRNA for 24 h and then infected with adenovirus encoding null or VEGFR2 for another 24 h (n = 5 independent experiments). Expression of VEGFR2 and FUNDC1 was determined by western blot assay. f Representative images of spheroid-sprouting are shown. Sprout length in ten random microscopic fields per group were measured by NIH ImageJ and statistically analyzed. (n = 5 independent experiments). Scale bar, 100 µm. g Matrigel containing vascular endothelial growth factor (VEGF) was injected subcutaneously into FUNDC1f/YCdh5− and FUNDC1f/YCdh5+ mice that had received intravenous injections of VEGFR2 adenoviruses 24 h before. After 10 days, matrigel plugs were removed for analysis of new vessel formation by histological and Hb assays (n = 5 mice/group) (top). Quantification of Hb extracted from Matrigel plugs from FUNDC1f/YCdh5− and FUNDC1f/YCdh5+ mice (n = 5 mice/group) (bottom). h Established LLC tumors approximately 90 mm3 in size were treated with either intravenous injection of recombinant VEGFR2 adenoviruses or Ad-Null as control (n = 5 mice/group). Scale bar, 100 µm. After 28 days, the tumors were harvested and quantified. Representative images of tumors were shown. i Immunostaining of LLC tumor sections and quantification of relative CD31-positive area. (n = 5 mice/group). Scale bar, 100 µm. Statistical significance was assessed using two-tailed t-tests for two groups and using one-way ANOVA with post hoc multiple comparisons test for multiple groups. *p < 0.05 vs Scr siRNA; Ad-Null or FUNDC1f/YCdh5−+Ad-Null. All values are mean ± S.D.