Hg3 31

Formalin-fixed and paraffin-embedded tissue samples of BC were cut into 4 μm sections. For the subsequent immunohistochemical staining, a BenchMark XT immunostainer (Ventana Medical Systems, Tucson, AZ) was used. For antigen retrieval, sections were incubated in CC1 mild buffer (Ventana Medical Systems, Tucson, AZ) for 30 min at 100 °C or in protease 1 for 8 min. The sections were stained with anti-p53 antibody (DO-7, Dako, 1:50), anti-GATA 3 antibody (HG3-31, SantaCruz, 1:50), anti-ER (SP1, Ventana, ready to use), anti-Her2neu (4B5, Ventana, ready to use), anti-CK5/6 (EP24,EP67, abcam, 1:100), anti-CD44 (DF1485, Dako, 1:50), anti-CK20 (KS20.8, Dako, 1:100) and anti-Uroplakin III (AU1, Progen, ready to use) for 60 min at room temperature, and visualized using the avidin–biotin complex method and DAB. A detailed description of the antibodies used for the study can be found in Additional file 1: Table S3. We stained the cell nuclei by additionally incubating for 12 min with hematoxylin and bluing reagent (Ventana Medical Systems, Tucson, AZ).
The stains were evaluated using an Olympus BX50 and Olympus BX46 microscopes (Olympus Europe). Histological images were acquired with the digital slide scanner PANNORAMIC 1000 (3DHISTECH).

T47D cells were grown in 150 mm tissue culture plates and fixed with 1% formaldehyde for 10 min at room temperature. After fixation, chromatin samples were obtained using the enzymatic shearing method (ChIP-IT Express, Active Motif). Samples were immunoprecipitated with 2 μg of antibody against GATA3 (HG3-31, Santa Cruz). Mouse IgG (Active Motif) was used as negative control. Primer sets for ChIP-qPCR were designed as described in (28 (link)) and are listed in Supplementary Table S3. Real-time qPCR was performed using SYBR Green (Applied Biosystems, API) to assess DNA-binding fold changes.

The antibodies were as follows: anti-APG5 (FL-275 Santa Cruz sc-33210, dilution 1:1,000) for WB, anti-LC3B (Sigma-Aldrich L7543, dilution 1:100) for WB and PLA, anti-p-Stat6 pY641 (BD Pharmingen 558241, dilution 1:1000) for WB, anti-Stat6 (Cell Signaling 9362, dilution 1:1000 for WB and 1:500 for PLA) for WB and PLA, anti-IRF4 (M-17 Santa Cruz sc-6059, dilution 1:1000 for WB and 1:500 for PLA) for WB and PLA, anti-GATA-3 (HG3-31 Santa Cruz sc-268, dilution 1:1000 for WB) for WB and anti-GATA-3 (H-48 Santa Cruz, dilution 1:500) for PLA, anti-PU.1 (T-21 Santa Cruz sc-352, 10 µg) for IP and (D-19 sc-5949, dilution 1:100) for PLA, anti-PU.1 (Cell Signaling 2266, dilution 1:100) for WB, anti-p62 (G962-C Progen, dilution 1:1000) for WB, mono- and polyubiquitinylated conjugates, mAb (FK2) HRP conjugate (Enzo BML-pw0150 dilution 1:1000) for WB, K48-linkage specific-polyubiquitin (D9D5 Cell Signaling 12805, dilution 1:1000) for PLA, K63-linkage specific-polyubiquitin (D7A11 Cell Signaling 12930, dilution 1:500) for PLA, anti-β-actin (Sigma-Aldrich, dilution 1:1000), Rabbit, Goat and Mouse IgG Isotype Control (Thermo Scientific, dilution 1:500 for PLA and 10 µg for IP) for IP and PLA, paxillin antibody B2 (Santa Cruz sc-365379, dilution 1:1000) and HDAC1 (Cell Signaling 2062, dilution 1:1000) for WB.