Ap 2γ

Cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 for 10 min, and blocked with 3% bovine serum albumin for 30 min at room temperature. After blocking, cells were incubated overnight at 4 °C with primary antibodies. Primary antibodies are NANOG, TRA-1-81 (1:250, Stem cells); KRT8, KRT18, KRT19, AP-2γ, FOXG1 (1:200, Abcam); ALDH1A3, AP-2α, AFP (1: 200, Santa Cruz); Brachyury (T) (1:50, Novus); P63 (1:100, Genetex); p-SMAD1/5/9 (1:200, Cell Signaling); CDH1, Desmoplakin (1:500, BD Biosciences). Alexa 594-conjugated secondary antibody (1:400, red, Molecular Probes, Eugene, OR) or an Alexa 488-conjugated secondary antibody (1:400, green, Molecular Probes) was used to visualize the staining. Following three washes with PBS, slides were mounted with the VECTASHIELD mounting medium (Vector Laboratories, Burlingame, CA). Prior to mounting, slides were incubated with 2 μM 4′,6-diamidino-2-phenylindole (DAPI) fluorescence (Molecular Probes) for 10 min at 37 °C to stain the nuclei. The fluorescence images were taken using the EVOS FL Auto Cell Imaging System fluorescence microscope (ThermoFisher Scientific, NY, USA).

Proteins were extracted from human breast cancer cells using RIPA lysis buffer (Sigma- Aldrich) and protein concentration was determined by the BCA Protein Assay Kit (Thermo). Proteins (20 μg) were separated on 4–20% gradient gels and transferred onto PVDF membranes using Trans-Blot Turbo transfer pack (Bio-Rad) and Trans-Blot Turbo transfer system (Bio-Rad). Membranes were blocked in Odyssey blocking buffer (LI-COR) and incubated with primary antibodies overnight at 4 °C. The primary antibodies were AFP, AP-2α, GATA3, ID1, ID2, and Actin (Santa Cruz); FOXA2, AP-2γ, SOX11, KRT8, and KRT18 (purchased from Abcam); Brachyury (T) (Novus); P63 (Genetex); OCT4, SOX2, and NANOG (purchased from Stem cells); p-SMAD1/5/9 (purchased from Cell Signaling); OTX2 (R&D Bioscience); TUJ1 (Promega Inc). Primary antibodies were used at 1:1000 dilution overnight at 4 °C. The membranes were then incubated with IRDye 680CW or IRDye 800CW secondary antibodies (1:50000, LI-COR) for 1 h at room temperature. The membranes were scanned using the Odyssey infrared imaging system (LI-COR).

Hippocampal DG of juvenile and adult AP2γ KO mice and WT littermates were carefully macrodissected out after occision. The tissue was weighted and homogenized in RIPA buffer [containing 50 mM Tris HCl, 2 mM EDTA, 250 mM NaCl, 10 % glycerol, 1 mM PMSF protease inhibitors (Roche, Switzerland)] and then sonicated (Sonics & Materials, US) for 2 min. Samples were centrifuged for 25 min at 10,000 rpm and 4 °C. The protein concentration of the supernatant was determined using Bradford assay. Samples with equal amounts of protein, 30 μg, were analyzed using the following primary antibodies: alpha-tubulin (#5168; Sigma, mouse, 1:5000), AP2γ (#31288; goat, 1:500; Abcam, UK), Pax6 (#2237; rabbit, 1:1000; Millipore, US), Sox2 (#7935; mouse, 1:500; Abcam, UK) and Tbr2 (#2283; rabbit, 1:500; Millipore, US). Secondary antibodies were used from BioRad (Anti-mouse, 1:10.000; #1706516; Anti-rabbit, 1:10.000; #1706515, US) and Santa-Cruz Biotechnologies (Anti-goat, 1:7500; #A2216, US). Membranes were developed using SuperSignal west Femto reagent (#34096; ThermoFisher, US) and developed in Sapphire Biomolecular Imager from Azure Biosystems (US). After developing, images were quantified using AzureSpot analysis software (Azure Biosystems, US).