Fig 2: Eya3 interacts directly with the PP2A-B55α subunit. a Top: GST-Aα, GST-Aα + B55α, GST-Aα + B55α + Cα, or His-B55α were used to pull down purified Flag-Eya3, which was identified using an anti-Flag antibody and western blot analysis. Bottom: Coomassie blue stained SDS PAGE gel demonstrating the amount of PP2A subunits used for pull-down experiments. b His-B55α and GST-B56γ1 were used to pull down purified Flag-Eya3, which is identified using an anti-Flag antibody and western blot analysis. Bottom: Coomassie blue stained SDS-PAGE gel demonstrating the amount of PP2A subunits used for pull-down experiments. c Western blot analysis shows protein levels of Flag-Eya3 and PP2A-B55α in SCR or PP2A-B55α KD HEK293FT cells transfected with Flag-Eya3. Molecular weight markers in kilodalton are labeled on the side of western blots. d Representative images of PLA demonstrate the Eya3–PP2A-C interaction in SCR or PP2A-B55α KD HEK293FT cells transfected with Eya3, scale bar: 10 μm. e Quantification of PLA/DAPI signal ratio in d, SCR group is set to 1 and p-value is calculated using an unpaired Student’s t-test, **p < 0.01, error bars: standard deviations (SD), n = 5. f Western blot analysis shows endogenous protein levels of Eya3 and PP2A-B55α in SCR, Eya3 KD, or PP2A-B55α KD 66cl4 cells. g Representative images of PLA demonstrate the Eya3–PP2A-C association in SCR, but not in Eya3 KD or PP2A-B55α KD 66cl4 cells, scale bar: 10 μm. h Quantification of PLA/DAPI signal ratio in g, SCR group is set to 1 and p-value is calculated using the one-way ANOVA followed by Tukey test, ***p < 0.001, error bars: SD, n = 5

Fig 3: The Eya3–PP2A association is disrupted by the Eya3 H79A mutation in cells, and can be observed in the endogenous context. a Western blot analysis on whole cell lysates demonstrates levels of exogenous Eya3, as detected with an anti-Eya3 antibody, expressed in HEK293FT cells transfected with empty vector (EV), Flag-Eya3WT or Flag-Eya3H79A. Molecular weight markers in kilodalton are labeled on the side of western blots. b Representative images of proximity ligation assay (PLA) demonstrating the association between Eya3 and PP2A-C in HEK293FT cells transfected with EV, Eya3WT, or Eya3H79A are shown, scale bar: 20 μm. c Quantification of relative PLA/DAPI signal ratio in b, EV group is set to 1 and p-value is calculated using one-way ANOVA followed by Tukey test, ***p < 0.001, error bars: standard deviations (SD), n = 5. d Representative PLA images examining the endogenous Eya3–PP2A-C association in multiple mouse and human breast cancer lines: 66cl4, Met1, MCF7, and MDA-MB-231 are shown, scale bar: 20 μm

Fig 4: All Eya family members interact with PP2A, and the N-terminal domain (NTD) of Eya3 is the main contributor to the PP2A interaction. a HEK293FT cells were transfected with EV, Flag-fused human Eya1 (hEya1), hEya2, hEya3, or hEya4. IP against Flag demonstrates the levels of PP2A-C subunit associated with Flag-hEyas. Molecular weight markers in kilodalton are labeled on the side of western blots. b Thr phosphatase activity assay was conducted using hEyas immunoprecipitated from transfected HEK293FT cells. All phosphatase activity was normalized to the amount of protein used in the assay. hEya3 Thr phosphatase activity was set to 100%. c HEK293FT cells were transfected with EV, Flag-fused wildtype (WT), NTD, or C-terminal domain (CTD) Eya3. IP against Flag demonstrates the levels of PP2A-C subunit associated with different domains of Flag-Eya3. d Thr phosphatase activity assay was conducted using different domains of Eya3 immunoprecipitated with a Flag antibody from transfected HEK293FT cells. Eya3 WT Thr phosphatase activity was set to 100%. Data shown are mean ± SD from three independent experiments. In the phosphatase activity assay, the p-value is calculated using one-way ANOVA followed by Tukey test, **p < 0.01, and ***p < 0.001

Fig 5: Eya3–PP2A-associated Thr phosphatase activity increases breast cancer metastasis and positively correlates with the percentage of c-Myc-positive regions in lung metastases. Experimental metastasis assay was performed by injecting BALB/c mice with firefly luciferase-tagged 66cl4 cells as follows: SCR + EV, KD + EV, KD + Eya3WT, or KD + Eya3H79A. a Representative bioluminescent images of metastatic burden in mice at week 3 after injection. b Quantification of photon flux in a, p-value is calculated using one-way ANOVA followed by Tukey test, *p < 0.05, **p < 0.01. c Representative immunohistochemical staining of lung metastases using a c-Myc antibody. Note that the percentage of c-Myc positive cells is highest in the presence of WT Eya3 (either endogenous or addback), and is diminished with Eya3 KD or addback of the Eya3H79A mutant. d Graph shows the quantification of c-Myc positive regions across different conditions. Percentage of positive cells was assessed using ImageJ software. p-value was calculated using one-way ANOVA followed by Tukey test, ***p < 0.001, error bars: standard deviation. 20 metastases from each condition were examined, scale bar: 20 μm. e Kaplan–Meier curves demonstrate the percentage of mice that survived until day 70. p-value is calculated using the log-rank test followed by Bonferroni correction, ***p < 0.001