Fig. 5 | Nature Communications

Fig. 5

From: A LATS biosensor screen identifies VEGFR as a regulator of the Hippo pathway in angiogenesis

Fig. 5

VEGFR regulates angiogenesis and tumor VM through YAP/TAZ in vitro. a Transient knockdown of YAP and/or TAZ in MCF10A overexpressing VEGFR2 decreases expression of ANG-2 and CYR61. Western blotting exposures indicate relative expression of YAP and TAZ. b, c VEGFR2 overexpression and VEGF treatment increases tube formation by MCF10A through YAP/TAZ. YAP and/or TAZ were transiently knocked down by siRNA in MCF10A stably overexpressing VEGFR2 and subjected to tube-formation assay on Matrigel 48 h after transfection alongside wild type MCF10A. For some conditions, cells were stimulated with 100 ng ml−1 VEGF or were treated with 100 nM verteporfin for the duration of the tube formation assay. Representative images are shown in b. Scale bar denotes 200 μm. Total tube formation was quantified in c (n = 3). *p < 0.05 in two-sample unpaired t-test. dl YAP/TAZ are critical for VEGF-induced angiogenesis in HUVEC (df) and BOEC (gi), as well as for vasculogenic mimicry in MDA-MB231 (jl). d, g, j YAP and/or TAZ were transiently knocked down by siRNA in each cell line which reduced ANG-2 and CYR61 expression. Western blotting exposures indicate relative expression of YAP and TAZ for each cell line. e, h, k Representative images of tube formation on Matrigel assessed 48 h after transfection. For some conditions, cells were stimulated with 100 ng ml−1 VEGF or were treated with 100 nM verteporfin for the duration of the tube formation assay. Scale bar denotes 200 μm. Total tube formation was quantified in f, i, l (n = 3). *p < 0.05 in two-sample unpaired t-test. m, n Exogenous CYR61 and ANG-2 can partially rescue tube formation in YAP/TAZ knockdown HUVEC. YAP and/or TAZ were transiently knocked down by siRNA in HUVEC. Tube formation on Matrigel was assessed 48 h after transfection. For some conditions, cells were stimulated with 100 ng ml−1 VEGF, 200 ng ml−1 CYR61, and/or 200 ng ml−1ANG-2 for the duration of the tube formation assay. Scale bar denotes 200 μm. Representative images are shown in m and total tube formation was quantified in n. (n = 3). All data are represented as mean ± SD

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