Figure 1.
Gata3 deficiency promotes basal differentiation and reduces proliferation in MECs, and p18 deficiency rescues proliferative defects caused by Gata3 heterozygosity. (A) MECs isolated from 3-month-old female Gata3f/f mice were infected with pMX-Cre (Cre) and pMX-Empty (Empty) then selected with puromycin for 3 days. mRNA was extracted and analyzed. (B, C) RNA and protein lysates extracted from WT and Gata3+/- MECs were analyzed by qRT-PCR (B) and western blot (C). Data in (A) and (B) represent the mean ± SD from triplicates of two independent primary cell lines of each genotype. The asterisk (*) denotes a statistical significance between WT and Gata3+/- or Cre and Empty samples as determined by T-test. (D) Representative mammary tissues from 8-10-month-old mice were analyzed by immunohistochemistry with Ki67. (E) The percentages of Ki67-positive cells were calculated from cells situated in clear duct/gland structures from 2-4 month and 8-10-month-old mice, respectively. Results represent the mean ± SD of three animals per group. (F-H) Tumor-free mammary glands (MG, F, G) and mammary epithelial cells (MEC, H) from 2-4-month-old mice were analyzed by western blot (F) and qRT-PCR (G, H). Data are expressed as the mean ± SD from triplicates of each of three separate mice (G) or of four different lines of MECs (H). The asterisk (*) denotes a statistical significance from p18+/-;Gata3+/- and p18+/- samples determined by T-test. (I) Representative mammary cells from 2-4-month-old mice were analyzed by flow cytometry. (J) Freshly isolated mammary cells from 2-4-month-old mice were cultured in Matrigel-coated 24 well plates. Nine days after culture, the colonies were immunostained with Ck8 and Ck5 (left panel). Ck5 expression in colonies was quantified by ImageJ software (right panel). The assay was performed in triplicate for each animal. The bar graphs represent the mean ± SD of two animals per group.