Gata3 d13c9

Tissue microarrays (1.0 mm cores, 4 μm sections) were analyzed with antibodies against CCNB1 (Y106, Epitomics), EGFR (3C6, Ventana), FOXA1 (2F83, Abcam), FOXM1 (C-20, Santa Cruz), GATA3 (D13C9, Cell Signaling), phospho-HISTH3 (Ser10) (#9701, Cell signaling), KRT5 (EP1601Y, Thermo Scientific), LAMA5 (4C7, Dako), PLK1 (208G4, Cell signaling), PPARG (C26H12 Cell Signaling), RXRA (F-1, Santa Cruz), STAT3 (124H6, Cell signaling), phospho-STAT3 (Tyr705) (D3A7, Cell signaling). Cores were evaluated as blinded digitalized image files. For quantitatively staining markers (EGFR, FOXA1, GATA3, KRT5, PPARG, RXRA, STAT3) a tumor cell score (TCS) was defined as described in Sjödahl et al. [4 (link)]. For discrete cellular labeling (CCNB1, FOXM1, PLK1, p-STAT3), fractions of positive tumor cells was recorded. The mean tumor cell score of core pairs from the same sample was calculated. The number of cores evaluated for each marker ranged from 480 to 524. Mitotic figures were identified by the phospho-HISTH3 (Ser10) antibody and basal lamina by anti-Laminin α-5 staining. When possible, lines were drawn through the plane of the mitotic figure (in the direction of the cell division), and tangentially along the nearest basal lamina (Photoshop CS5 version 12.1), and the distance, in cell layers, to the basal membrane recorded. A total of 416 mitoses from 112 different tumors were analyzed.

Cells were lysed in RIPA buffer containing PMSF and protease and phosphatase inhibitors (Santa Cruz Biotechnology) for 30 minutes on ice. Proteins were separated on denaturing 4%–15% SDS-PAGE (Bio-Rad), transferred onto PVDF membrane (MilliporeSigma), and probed with Abs to GATA3 (D13C9), RUNX3 (D6E2), and β-actin (13E5, Cell Signaling Technology). Membranes were developed using HRP-conjugated secondary Abs and Pierce ECL Western blotting substrate (Thermo Fisher Scientific).

For immunofluorescence microscopy, cells were plated onto coverslips (VWR) in a 24-well plate. Next day, cells were washed twice with ice-cold PBS and fixed with 4% PFA +0.1% Triton X-100 in PBS for 20 minutes on ice. Cells were permeabilised with 0.5% Triton X-100 in PBS for 20 minutes and blocked with 10% FCS +0.1% Triton X-100 in PBS for 1 hour with three washes between individual steps. Primary (GATA3 D13C9, Cell Signaling #5852, 1:100) and secondary (AlexaFluor 546 goat anti-rabbit, Invitrogen, 1:500) antibodies were diluted in blocking solution and incubated for 1 hour at RT. Finally, cells were stained with DAPI (10μg/ml, Sigma) for 10 minutes at RT in the dark. Slides were mounted in 85% glycerol and images were acquired on a Zeiss LSM 710 confocal imaging system.

Whole cell lysates were obtained using 8 M urea lysis buffer (8 M urea, 1% SDS, 0.125 M Tris, pH 6.8). Protein extracts (15 μg) were resolved on SDS–PAGE gels and immunoblotted using the following antibodies: GATA3 (D13C9; Cell Signaling Technology, Danvers, MA), FOXA1 (ab23738; Abcam, Cambridge, MA, USA), ERα (sc-543; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and actin (ab8226; Abcam). Signal intensity was analyzed using rectangular volume tool in Quantity One Analysis Software (Bio-Rad, Hercules, CA, USA) with global background subtraction.

Immunostaining of TERT was performed using rabbit monoclonal to telomerase reverse transcriptase antibody (Y182) (Abcam). This monoclonal antibody was prepared using a synthetic peptide of the human telomerase reverse transcriptase aa1100‐1200 (C‐terminal) as an immunogen. The conditions for immunostaining were performed as follows. Antigen retrieval was performed by heat mediation at 95°C for 20 min using a microwave. Samples were incubated with primary antibody (1/100) for 30 min. An undiluted HRP‐conjugated goat anti‐rabbit IgG polyclonal was used as the secondary antibody. Tumor cells with more than 30% staining were considered positive in the current study. Immunohistochemical investigation of TBX21 (4B10; Abcam) and GATA3 (D13C9; Cell Signaling Technology) was performed for PTCL‐NOS. Tumor cells with more than 30% staining were considered positive as in our previous study.²⁴