Nuclear localization of Stat5a (Nuc-Stat5a) is lost during breast cancer progression. (A) Detection of nuclear-localized Stat5a in Material I with immunofluorescence and quantified with AQUA revealed a significant reduction in Nuc-Stat5a protein in invasive ductal carcinoma (IDC; n = 66) and lymph node metastases (n = 19) when compared with normal breast tissue (n = 23) and ductal carcinoma in situ (DCIS; n = 18). (B) Levels of nuclear localized Stat5b (Nuc-Stat5b) remained unchanged between normal (n = 24), DCIS (n = 12), IDC (n = 67), and lymph node metastases (n = 13) in the same breast progression array (Material I). (C) Representative images of Stat5a, Stat5b, and pY-Stat5a/b detected with immunofluorescence in normal human breast tissue and lymph node metastases (Material I). Stat5a, Stat5b, pY-Stat5a/b, red (Cy5); cytokeratin, green (FITC); nuclei, blue (DAPI). (D) Stat5a protein (upper panels) translocated to the nucleus after ex vivo prolactin stimulation of human breast tissue explants, whereas prolactin induced only minimal nuclear localization of Stat5b protein (middle panels). Corresponding with enrichment of nuclear Stat5a protein in response to prolactin, tyrosine-phosphorylated Stat5a/b (pY-Stat5a/b) was induced in the nuclei of the same breast tissue after ex vivo stimulation with prolactin (lower panels). (E, F) Levels of total cellular Stat5a protein (E), but not total cellular Stat5b protein (F), were significantly reduced over breast cancer progression (Material I). (G, H) Stat5a mRNA expression levels in human breast tumor tissue (Material II) were associated with relapse-free survival (G), whereas Stat5b mRNA expression levels were not associated with relapse-free survival (H). Statistical differences (ANOVA, Dunnett T3 post hoc test) in levels of nuclear and total cellular Stat5a or Stat5b over breast cancer progression in relation to normal histologic type are indicated. ***p < 0.001; *p < 0.05. AQUA, Automated Quantitative Analysis; DCIS, ductal carcinoma in situ; IDC, invasive ductal carcinoma; IQR, interquartile range; LN Met, lymph node metastasis.