Foxa2

Cells were rinsed in PBS and fixed in 4%PFA for 15 mins. Cells were then permeabilised via three washes in PBS containing 0.3% Triton-X-100 (PBST) and then blocked in PBST containing 1% BSA and 3% normal donkey serum. Primary antibodies were added in blocking solution for 2 hours at ambient temperature or overnight at 4 °C. The cells were washed in PBST three times before being incubated for 1 hour in the dark in Alexa-Fluor secondary antibodies, 1:200 (Invitrogen). Three PBST washes were then performed that included one with DAPI, 1:1000 (Molecular Probes). The primary antibodies used in this study were: FolR1(sheep, R&D), TH (rabbit, PelFreez), Pitx3 (rabbit, gift of M. Smidt, University of Amsterdam), Foxa2 (goat, Santa Cruz), Foxa2 (Rabbit, Abcam), Lmx1a (rabbit, gift of M. German, UCSF), Nestin (mouse, BD Pharmagen), GFAP (rabbit, Dako), GABA(rabbit, Sigma), 5HT(mouse, Abcam), Isl1(mouse, DSHB), Lim1/2(mouse, DSHB), Pax6(mouse, DSHB), Nkx6.1(mouse, DSHB), Dmrt5(rabbit, custom made). Images were taken on a Leica TCS SP5 confocal microscope. Quantification of markers was carried out manually by examining randomly selected fields from at least 3 independent experiments and presented as means ± sem.

Cells cultured on 6-well plates (1000 μL/well)/12-well plates (400 μL/well) were divided into different groups. After washing with phosphate-buffered saline + 0.05% Tween (PBST) three times (5 min each time), the cells were fixed with precooled methyl alcohol for 20 min at − 20 °C. Triton X-100 (0.5%) was added to the cell slide and incubated for 20 min for transparency. After that, the cells were washed with PBST and blocked with bovine serum albumin (BSA) (Abcone) for 30 min. Finally, the cells were incubated overnight with SOX17 (Rabbit, Proteintech, 1:100)/FOXA2 (Mouse, Abcam, 1:200), HNF4A (Rabbit, Abcam, 1:200)/AFP (Mouse, ABclonal, 1:100), CK19 (Rabbit, Proteintech, 1:100)/ALB (Mouse, Proteintech, 1:100), or β-catenin antibody (Rabbit, Proteintech, 1:100) at 4 °C. After washing 3 times, the cells were incubated with goat anti-mouse/rabbit IgG-FITC or goat anti-mouse/rabbit IgG-RBITC fluorescent secondary antibodies (Earthox, 1:500) for 60 min. Then, the cells were mixed with DAPI reagent for 10 min. Cell slides were observed under an optical microscope (MF52-N, Mshot) or with Axio Imager M2 software (Nikon) (×100, ×200 magnification).

The tissue specimens were fixed overnight in 10% neutral buffered formalin and then dehydrated in increasing concentrations of ethyl alcohol, followed by clearing of alcohol by xylene. Uterine slices were deparaffinized and incubated in citrate buffer for antigen retrieval by hyperbaric heating and then incubated overnight at 4 °C with the primary antibodies including Menin (Bethyl, 1:1000), COX2 (Santa Cruz, 1:200), Dtprp (homemade, 1:200), Ki67 (Servicebio,1:200), PL1 (Santa Cruz, 1:200), 3β-HSD (Santa Cruz, 1:200), p450scc (Santa Cruz, 1:200), PR (Cell Signaling Technology, 1:200), HAND2 (Santa Cruz, 1:200), p27 (Abcam,1:100), pH3 (Cell Signaling Technology, 1:200), ERK1/2 (Cell Signaling Technology, 1:200) and p-ERK1/2 (Cell Signaling Technology, 1:200). A Histostain-SP Kit (Zhongshan Golden Bridge Biotechnology) was applied to visualize the antigen. Immunofluorescence staining for OCT4 (Cell Signaling Technology, 1:200), β-Catenin (Abcam, 1:200), H3K27me3 (Cell Signaling Technology, 1:200), BrdU (Abcam, 1:500), PCNA (Santa Cruz, 1:200) and FOXA2 (Abcam, 1:500) was performed in paraffin-fixed sections and secondary antibody Cy^TM3 AffiniPure Goat Anti-Rabbit IgG(H + L) (Jackson ImmunoResearch,1:200) were used. The images were captured by Leica DM2500 light microscope. Antibodies with detailed information are listed in Supplementary Table 1.