Fig. 4 | Nature Communications

Fig. 4

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

Fig. 4

VEGFR is an upstream regulator of Hippo signaling. VEGFR inhibition activates LATS-BS a and suppresses YAP/TAZ transcriptional co-activation in HEK293A b. Cells were treated with each inhibitor for 4 h at 10 μM (n = 3). c VEGFR inhibition diminishes expression of YAP/TAZ targets, CYR61/CTGF. HEK293A were treated with inhibitors for 4 h at 10 μM. CYR61 or CTGF mRNA expression was determined by qRT-PCR (n = 3). d VEGFR reduces YAP-S127 phosphorylation in HEK293 (western blotting). e VEGF stimulation inhibits LATS-BS activity in MCF10A stably overexpressing VEGFR1/2. MCF10A-VEGFR1/2 were transfected with LATS-BS. Cells were treated with VEGF (100 ng ml−1) for the indicated times. For some samples, cells were pre-treated with axitinib at 10 μM for 3 h before VEGF treatment (n = 3). f, g VEGF increases YAP/TAZ transcriptional co-activation of CYR61 in MCF10A-VEGFR1/2 (f), as well as in BOEC and MDA-MB231 (g). Cells were treated with 100 ng ml−1 VEGF for the indicated times. CYR61 expression was measured by qRT-PCR. For some samples, cells were pre-treated with Axitinib at 10 μM for 3 h before VEGF treatment (n = 3). h-m VEGF stimulation increases YAP/TAZ nuclear localization in MCF10A-VEGFR2 (h, i), MDA-MB231 (j, k), and BOEC (l, m). h, j, l Representative images of YAP or TAZ immunostaining are shown after treatment with 100 ng ml−1 VEGF for the indicated times. Scale bar represents 15 μm. i, k, m YAP/TAZ subcellular localization was quantified in three separate experiments in which at least 200 cells were examined. n, o VEGFR2 signals through PI3K, AKT, and MEK to inhibit LATS-BS (n) and activate the STBS reporter (o). Cells were untreated or treated with VEGFR inhibitor (axitinib), PI3K inhibitor (LY294002), AKT inhibitor (triciribine), or MEK inhibitor (PD98059) at 10 μM for 4 h (n = 3). *p < 0.05 in two-sample unpaired t-test. p VEGF stimulates phosphorylation of VEGFR, AKT, and ERK in HUVEC cells, and reduces MST1 and LATS1 phosphorylation (western blotting). HUVEC cells were treated with 100 ng ml−1 VEGF for the indicated times. VEGFR was inhibited using 10 μM axitinib for 3 h before VEGF treatment. All data are represented as mean ± SD

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