FIGURE SUMMARY
Title

The EYA Tyrosine Phosphatase Activity Is Pro-Angiogenic and Is Inhibited by Benzbromarone

Authors
Tadjuidje, E., Wang, T.S., Pandey, R.N., Sumanas, S., Lang, R.A., and Hegde, R.S.
Source
Full text @ PLoS One

EYA3 knockdown in HUVECs attenuates single cell motility and capillary morphogenesis.

(a) Expression of Eya transcripts in HUVECs. Real-time PCR product was analyzed at saturation (cycle 45) on a 1.5% agarose gel to confirm that amplified products were of the expected sizes. Indicated are the numbers of amplification cycles after which each signal was detectable; EYA4 was not detected (ND) after 45 cycles of amplification. RT- stands for cDNA from a reaction without reverse transcriptase. Graph below show the results of quantitative RT-PCR on HUVECs transduced with lentivirus scramble control RNA or shEYA3. (b) Change in cell density of HUVECs-scramble control and HUVEC-shEYA3 after 24 hours shows a small but significant increase when EYA3 levels are reduced. (c) Transwell migration of HUVEC-scramble control and HUVEC-shEYA3 shows a significant reduction in motility when EYA3 levels are reduced. (d) Capillary morphogenesis on matrigel was assayed using HUVEC-scramble control and HUVEC-shEYA3. A significant reduction in tube formation was seen when EYA3 levels were lowered. In each case the bars represent the mean and standard error of three experiments. The p-values were calculated by one-way ANOVA followed by a Tukey post test. ns is not significant, * p<0.05, ** p<0.01, *** p<0.001. In each case the p value shown is relative to the scramble-control.

EYA inhibitor Benzbromarone, compound 1.

(a) Compound 1 was identified from a screen of the NCI Diversity Set II library. IC50 values for compound 1 were measured for Eya3(ED), human EYA3 and human EYA2(ED) using as substrates the phosphotyrosine mimic pNPP and a phosphopeptide representing the last 10 amino acids of H2AX, a known EYA substrate. (b) Substrate titration shows that compound 1 is an uncompetitive inhibitor of EYA3(ED). Increasing concentration of substrate does not overcome inhibition. Each point represents the mean and standard deviation of two independent readings. (c) Plots of Vmax and Km as a function of inhibitor concentration show that both values decrease with increased inhibitor concentration. Values in (c) were derived from nonlinear regression analyses of curves in (b) using PRISM (GraphPad Software). (d) Compound 1 does not affect the interaction between EYA3 and SIX2. Recombinant purified EYA3 and His-SIX2 were mixed and treated with either the vehicle control (1% DMSO) (lane 1) or 50 µM compound 1 (lane 5) for 15 minutes at room temperature. The mixture was loaded on a Ni-NTA column. Beads were washed with 3 column volumes of load buffer (last washes, lanes 2 and 6). Proteins retained on the beads are shown in lanes 3 and 7. Lanes 4 and 8 are molecular weight markers.

EYA inhibitors attenuate migration and tubulogenesis of HUVECs.

(a) Percent migration of HUVECs in the presence of 5 μM relative to the vehicle control?. (b) Change in cell density after 24 hours in the presence of either vehicle control (0.1% DMSO) or 5 μM of each EYA inhibitor. (c) Quantitation of the number of tube-like structures formed by HUVECs in the presence of either the vehicle control (0.1% DMSO) or the indicated concentrations of compounds 1, 1a and 1b. The number of tubes was measured using NeuroJ. Data are mean and standard error of three independent experiments. p-values from a one-way ANOVA are shown; ns is not significant, * p<0.05, ** p<0.01, *** p<0.001. (d) Representative images of HUVECs on Matrigel in the presence of the indicated doses of 1a.

EYA inhibitors attenuate sprouting angiogenesis.

(a) Representative images of aortic rings treated with either the vector control (0.1% DMSO) or 5 μM of compounds 1, 1a, or 1b. Rings were stained with isolectin. 1c is used as a negative control as it does not inhibit EYA. (b) Quantitation of the number of sprouts per ring is included; each bar represents the mean of 6 rings. p-values from a one-way ANOVA are shown; ns is not significant, * p<0.05, ** p<0.01, *** p<0.001. (c) Compounds 1, 1a, and 1b in the indicated doses were used in aortic ring experiments. The number of sprouts per ring is plotted indicating that inhibition of aortic sprouting was dose-dependent.

Dose-dependent effects of EYA inhibitors on the developing zebrafish vasculature.

(a) Titration of compounds 1, 1a, 1b at the indicated doses. Two independent experiments were performed in most cases using 9–15 embryos per experiment at each dose; the standard error and mean values are shown. Experiments with 0.25 μM compound 1 and 5 μM compound 1b were only performed once using 10 embryos each. The y-axis shows the percentage of embryos showing any defects in vascular development (either intersegmental vessels (ISV) formation or defects in the dorsal aorta/cardinal vein). (b) Images of representative untreated control (CTL), vehicle (DMSO) treated, and EYA inhibitor treated embryos at 28–30 hpf. Note reduced or absent ISV in the inhibitor treated embryos relative to the controls (ISV in controls indicated by white arrows).

Acknowledgments
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