Fig 2: EYA3 PTP promotes survival of PASMC after DNA damage. In each experiment cells were treated with 200 µM H2O2 for 1 h. H2O2 was then withdrawn and the cells allowed to recover in normal culture medium. The percentage of viable cells (relative to untreated controls) were monitored using the WST-8 cell viability assay and are plotted versus time (x-axis). -1 h indicates the start of H2O2 treatment. H2O2 was withdrawn at 0 h. Each experiment was conducted at least three times. Representative data shown as the mean ± SD; statistical significance was determined using two-way ANOVA and Bonferroni’s post-test, ***P < 0.001, *P < 0.05. a Recovery from H2O2 treatment of control PASMC and PAH-PASMC (L10 and L85) shows that PAH-PASMC rapidly recover relative to normal PASMC. b EYA-PTP inhibition with Benzarone makes PAH-PASMC susceptible to H2O2-induced DNA damage, while having no effect on control PASMC. c Stable knockdown of EYA3 in PAH-PASMC L10 cells increases their susceptibility to H2O2 treatment. d Stable knockdown of EYA3 in PAH-PASMC L85 cells increases their susceptibility to H2O2 treatment. e EYA-PTP inhibition with 7.5 µM BZ has no effect on the susceptibility of PAH-PASMC L10-shEYA3 cells to H2O2 treatment. f EYA-PTP inhibition with 7.5 µM BZ has no effect on the susceptibility of PAH-PASMC L85-shEYA3 cells to H2O2 treatment. Source Data are provided as a Source Data file

Fig 3: The assembly of EYA3-SIX5-p300 complex in vivo and in vitro. (A) EYA3 pulled down both SIX5 and p300 in cancerous biopsies. Three independent cancerous were mixed with equal weights and were then immunoprecipitated using anti-EYA3 (or IgG, negative control) conjugated agarose. The output complexes were probed with antibodies as indicated in the figure. (B) EYA3 pulled down both SIX5 and p300 in HCEC-1CT cells expressing pCDNA3-Flag-EYA3. (C,D) Detection of direct interaction of EYA3-p300, EYA3-SIX5, p300-SIX5, and p300-EYA3 in vitro. Different combinations of plasmids as shown in the figure were transfected into HCEC-1CT cells, followed by immunoprecipitation with anti-Flag- and anti-Myc-agarose. The outputs were probed using anti-Flag and anti-Myc. (C) Interactions of EYA3-p300 and EYA3-SIX5. (D) Interactions of p300-SIX5 and p300-EYA3. (E) Schematic assembly of the EYA3-SIX5-p300 complex. IgG, immunoglobulin G.

Fig 4: Genetic loss of EYA3-PTP activity attenuates hypoxia-induced PH. a Right ventricular systolic pressure (RVSP) measured after the Sugen-hypoxia (Su-Hx) protocol is reduced in Eya3D262N mice relative to Eya3+/+ control animals. RVSP was measured upon return to room air. Mice maintained in room air served as controls (CTL). Two independent Su-Hx experiments were conducted with a total of 21 C57BL/6J Eya3+/+ and 18 Eya3D262N mice. RVSP measurements reported here represent successful catheterization and are plotted as the mean ± SD. Statistical significance was assessed using pairwise t-tests. b Right ventricular hypertrophy measured after the Su-Hx protocol is reduced in Eya3D262N mice relative to Eya3+/+ control animals. Right ventricular hypertrophy (Fulton’s index = right ventricular (RV) weight/left ventricle (LV) + interventricular septum weight) measured upon return to room air after the Su-Hx protocol, and in mice maintained in room air for an equivalent period. Data plotted as the mean ± SD. Statistical significance was assessed using pairwise t-tests. c Quantitation of the percentage of Ki-67-positive cells. Each data point represents at least six sections per lung. For all groups n = 4. Data plotted as the mean ± SD, statistical significance determined by pairwise t-tests. d Cell proliferation and muscularization is reduced in Eya3D262N mice subject to the Su-Hx protocol. Lung sections were stained with antibodies to a-SMA and Ki-67, and with the nuclear marker DAPI. Scale bar = 100 µm. e Genetic loss of EYA3-PTP activity reduces muscularization of DPAs but does not affect the endothelial cell layer. Representative sections from lungs of Eya3+/+ and Eya3D262N mice either maintained in room air or subject to the Su-Hx protocol were stained for the smooth muscle cell marker a-SMA (green), the endothelial cell marker von Willebrand factor (vWF; red), and the nuclear marker DAPI (blue). Scale bar = 100 µm. Source Data are provided as a Source Data file

Fig 5: Tyrosine phosphorylation of various EYA3 constructs, co-transfected with c-Src Y527F in HEK293T cells. (a) EYA3 tyrosine-phosphorylation quantified for EYA3 D309N constructs containing up to six Y?F mutations. (b), (c) Quantification of EYA3 tyrosine-phosphorylation performed with the purpose of evaluating the relative contribution to the total EYA3 phosphorylation of the residues mutated in the EYA3 T4 mutant. C-Myc-EYA3 proteins were co-expressed with c-Src Y527F in HEK293T cells. After Western blot verification of the lysates, the EYA3 proteins were immunoprecipitated using anti-c-Myc antibody. Then the samples were verified by a Western blot. For each immunoprecipitated EYA3 construct, the intensity of the band obtained from the anti-pY Western blot was divided by the one from the anti-c-Myc Western blot. Then, every value obtained was normalized to the value corresponding to EYA3 D309N. Bar graphs represent values of the quantified phosphorylation of the samples normalized to the quantified phosphorylation of EYA3 D309N. Quantification of the Western blot bands was conducted using ImageJ software [45]. For each of the experiments described (a, b, c), the values represent the mean ± SD of three independent experiments (n = 3) with statistics (unpaired t-test, two-tailed) (n.s. for not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).