(A) H&E staining of 8-week-old MMTV-Cre; GATA-3^flox/flox and MMTV-Cre; GATA-3^flox/+ mammary glands. fl/fl, GATA-3^flox/flox and fl/+, GATA-3^flox/+in (A) and (B).
(B) Immunofluorescence for GATA-3 (red), GFP (green), and DAPI (blue) in 8-week-old MMTV-Cre; GATA-3^flox/flox and MMTV-Cre; GATA-3^flox/+ mammary glands. White arrowhead denotes missing luminal epithelium and yellow arrowhead denotes GATA-3 null cells in ductal lumen.
Scale bars represent 25 μm in (A) and (B).
(C) PCR analysis of wild-type (WT) GATA-3, floxed (Fl) GATA-3, and deleted (Del) GATA-3 loci in 30-week-old MMTV-Cre; GATA-3^flox/flox, MMTV-Cre; GATA-3^flox/+, and Non-Tg; GATA-3^flox/+ mammary glands. Genomic DNA from the GFP+ (recombined) cell population of 30-week-old MMTV-Cre; GATA-3^flox/flox and MMTV-Cre; GATA-3^flox/+ was purified for PCR analysis. Whole-mammary epithelial DNA was used for Non-Tg; GATA-3^flox/+ analysis.

(A)GFP mammary whole mounts from 12-week-old WAP-rtTA-Cre; GATA-3^flox/+ and WAP-rtTA-Cre; GATA-3^flox/flox mice treated for 3 days (n = 5 flox/+, n = 4 flox/flox), 5 days (n = 4 flox/+, n = 5 flox/flox), and 14 days (n = 4 flox/+, n = 4 flox/flox) with doxycycline (dox). Cre, WAP-rtTA-Cre; fl/fl, GATA-3^flox/flox; and fl/+, GATA-3^flox/+ in (A)–(C).
(B and C) H&E staining (B) and immunofluorescence (C) for GATA-3 (red), GFP (green), and DAPI (blue) in 12-week-old WAP-rtTA-Cre; GATA-3^flox/+ and WAP-rtTA-Cre; GATA-3^flox/flox mice treated for 5 days and 14 days with doxycycline. Arrowheads in (C) denote GFP-positive/GATA-3-negative cells in ductal lumen.
Scale bars correspond to 3 mm in (A) and 25 μm in (B) and (C).

(A–H) Carmine-stained, number 4 mammary glands from 19-day-old to 8-week-old MMTV-Cre; GATA-3^flox/flox (n = 20) and MMTV-Cre; GATA-3^flox/+ (n = 18) mice. Shown are representative mammary glands for each time point (n = 4–6 mammary glands per time point). fl/fl, GATA-3^flox/flox and fl/+, GATA-3^flox/+ in (A)–(I). Scale bar corresponds to 3 mm.
(I) Ductal invasion in MMTV-Cre; GATA-3^flox/flox, MMTV-Cre; GATA-3^flox/+, and nontransgenic (Non-Tg); GATA-3^flox/+ mammary glands, represented as distance from proximal end of primary duct to distal TEB. Data are represented as mean ± SEM, n = 4–6 mammary glands per condition, *p < 0.01 (Student’s t test).

(A) Schematic representation of the microarray profiling strategy. Top, number 4 mammary gland of a 5-week-old β-actin-GFP mouse. TEBs, epithelium-free distal stroma (Stroma), and mature ductal epithelium (Mature duct) regions were microdissected for microarray profiling. Modified from Kouros-Mehr and Werb (2006)
(B and C) Immunofluorescence for GATA-3 (red) and DAPI (blue) in TEBs (B) and mature ducts (C) of 5-week-old mammary glands. Arrowheads denote cap cell layer of distal TEB. (D) Costaining for GATA-3 (green), α-smooth muscle actin (SMA, red), and DAPI (blue) in 12-week-old wild-type mammary glands. Arrowheads denote myoepithelial cells.
Scale bars correspond to 1 mm in (A) and 25 μm in (B)–(D).

(A–D) Immunofluorescence of mammary glands from 12-week-old WAP-rtTA-Cre; GATA-3^flox/+ and WAP-rtTA-Cre; GATA-3^flox/flox mice treated with doxycycline for 5 days. Antibodies used are as follows: (A) keratin 14 (K14, red) and keratin 18 (K18, green), (B) β-casein (green), (C) GATA-3 (red) and E-cadherin (E-cad, green), and (D) estrogen receptor (ER, red) and GFP (green). Sections were counterstained with DAPI (blue). Cre, WAP-rtTA-Cre; fl/fl, GATA-3^flox/flox; and fl/+, GATA-3^flox/+ in (A)–(G).
(E) Microarray profiling of whole mammary glands from 12-week-old WAP-rtTA-Cre;GATA-3^flox/+ and WAP-rtTA-Cre; GATA-3^flox/flox mice (n = 2) treated with doxycycline for 5 days. Data are represented as M = log₂(Het /Null) where Het = fluorescence intensity of WAP-rtTA-Cre; GATA-3^flox/+ sample and Null = fluorescence intensity of WAP-rtTA-Cre; GATA-3^flox/flox sample.
(F) PCNA immunostaining of 5 day doxycycline-treated WAP-rtTA-Cre; GATA-3^flox/+ (n = 4) and WAP-rtTA-Cre; GATA-3^flox/flox (n = 5) mammary glands. Scale bars correspond to 25 μm in (A)–(D) and (F).
(G) BrdU analysis of 5 day doxycycline-treated WAP-rtTA-Cre; GATA-3^flox/+ and WAP-rtTA-Cre; GATA-3^flox/flox mammary glands. Data are represented as the number of BrdU-positive cells per mammary duct; n = 50 ducts per condition, and error bars indicate mean ± SEM. ***p < 0.001 (Student’s t test).

(A–C) Immunofluorescence of mammary glands from 12-week-old WAP-rtTA-Cre; GATA-3^flox/flox mice treated with doxycycline for 14 days. Control mammary glands are similar to those shown in Figure 5. Antibodies used are as follows: (A) keratin 14 (K14, red) and keratin 18 (K18, green), (B) GATA-3 (red) and E-cadherin (E-cad, green), and (C) estrogen receptor (ER, red) and GFP (green). Sections were counterstained with DAPI (blue).
(D) Caspase-cleaved keratin 18 immunostaining of 5 day and 14 day doxycycline-treated WAP-rtTA-Cre; GATA-3^flox/flox and WAP-rtTA-Cre; GATA-3^flox/+ mammary glands (age = 12 weeks). Cre, WAP-rtTA-Cre; fl/fl, GATA-3^flox/flox; and fl/+, GATA-3^flox/+ in (D)–(H). Fourteen day dox-treated control mammary glands were similar to 5 day treated controls in (D) and (E).
(E) Quantification of caspase-cleaved keratin 18 (M30) expression from (D). Data are represented as the percentage of mammary ducts that contained at least one M30-positive cell; n = 10 ducts per condition.
(F) Carmine-stained whole mounts and GFP whole mounts of number 4 mammary glands from lactating WAP-rtTA-Cre; GATA-3^flox/flox (n = 2) and WAP-rtTA-Cre; GATA-3^flox/+ (n = 2) mice treated with doxycycline for 5 weeks. Tissues were harvested at day 10 of lactation. Corresponding H&E-stained sections are shown in (G).
(H) Weight of pups (in grams) born to 5 week doxycycline-treated WAP-rtTA-Cre; GATA-3^flox/flox and WAP-rtTA-Cre; GATA-3^flox/+ mothers at day 10 of lactation. Data are represented as mean ± SEM. *p < 0.01 (Student’s t test).
(I) PCR analysis of the FOXA1 promoter in adult mammary gland DNA immunoprecipitated with a GATA-3 antibody, control mouse immunoglobulin, or no primary antibody control. The PCR product was a 200 bp fragment 0.8 kb upstream of the FOXA1 promoter that contained a GATA binding site.