FIGURE 4.

Zfhx3 is necessary for prolactin-induced phosphorylation of STAT5. A, parental HC11 cells were treated with 5 μg/ml prolactin in a time-dependent manner and Stat5 phosphorylation at tyrosine 694 (p-Y694-Stat5) was detected by Western blotting. B, mouse HC11 mammary epithelial cells with stable knockdown of Zfhx3 (shZfhx3-8) or control (shCtrl) were treated with insulin (I), insulin plus dexamethasone (DI), or DIP for 7.5 min or 2.5 h and then subjected to Western blotting to detect p-Y694-Stat5 and total Stat5. C and D, stable knockdown of ZFHX3 in human breast cancer cell line T-47D also reduced prolactin (Prl)-induced STAT5 phosphorylation, as detected by Western blotting. Knockdown efficiency was confirmed by RT-PCR and Western blotting (D). E and F, IHC staining of p-Y694-Stat5 in lactation day 2 mammary glands (E). IHC score is shown as means ± S.E. (F) and was produced by multiplying the percentage of positive cells (scored as 0 for negative, 1 for < 10%, 2 for 11–50%, 3 for 50–80%, and 4 for > 80%) with the intensity of staining (0 for negative, 1 for weak, 2 for moderate, and 3 for strong). Statistical significance was calculated by one-way analysis of variance followed by Bonferroni's test. *, p < 0.05; **, p < 0.01. All Western blots were quantified using the ImageJ program.