Brachyury

Antibodies against octamer-binding transcription factor 4 (1:1000, Oct-4; ab200834, abcam), Brachyury (1:1000, sc-166962, Santa Cruz Biotechnology), GATA-binding protein 4 (1:1000, GATA4; sc-25310, Santa Cruz Biotechnology), SRY box transcription factor 17 (1:1000, Sox17; AF1924, R&D system), neuronal differentiation 1 (1:1000, NeuroD1; ab205300, abcam), XRCC5 (1:2000, AF5819, R&D system), IGF2BP1 (1:1000, ab184305, abcam), RBBP4 (1:1000, MAB7416, R&D system), NPM1 (1:1000, sc-271737, Santa Cruz Biotechnology), PCNA (1:1000, sc-56, Santa Cruz Biotechnology), GNAI2 (1:1000, ab137050, abcam), PHB2 (1:1000, sc-133084, Santa Cruz Biotechnology), LAMB1 (1:1000, sc-17810, Santa Cruz Biotechnology), LAMC1 (1:1000, sc-17751, Santa Cruz Biotechnology), β-catenin (1:1000, sc-7963, Santa Cruz Biotechnology), AKT (1:2000, #4691, Cell Signaling Technology), Jun amino-terminal kinases (JNK) (1:1000, #9252, Cell Signaling Technology), phosphorylated (p)-JNK (1:1000, #9255, Cell Signaling Technology), p38 (1:1000, sc-81621, Santa Cruz Biotechnology), p-p38 (1:1000, #9216, Cell Signaling Technology), TGF-β (1:1000, #3711, Cell Signaling Technology), CTGF (1:1000, sc-385970, Santa Cruz Biotechnology), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; (1:3000, sc-47724, Santa Cruz Biotechnology) were employed for the western blot analysis.

Cell clones were picked and cultured under feeder-free conditions in embryoid body stander medium (Millipore, Billerica MA, USA) to develop into embryoid bodies. The embryoid bodies were then transferred to gelatin-coated plates for further spontaneous differentiation for 5 to 10 days. Cells were then fixed with 4% paraformaldehyde for 30 minutes and permeabilized for an additional 10 minutes with 0.1% Triton X-100 (Sigma, St. Louis,MO,USA). The blocking step was 30 minutes with 2% fetal bovine serum in phosphate-buffered saline (PBS). Cells were incubated with antibody against nestin (mouse anti-human, 1:200; Santa Cruze Biotechnology,Santa Cruze,CA,USA), anti Sox-17 (rabbit anti-human, 1:200; Santa Cruz), and brachyury (mouse anti-human, 1:200; Santa Cruz) for 2 hours. Each antibody was detected using corresponding secondary antibodies conjugated to fluorescein isothiocyanate.

FFPE blocks were sectioned at 4 μm and a positive control was added to each slide. All IHC staining was fully calibrated on a Benchmark XT staining module (Ventana Medical Systems Inc., USA). Briefly, after sections were dewaxed and rehydrated, a CC1 Standard Benchmark XT pretreatment for antigen retrieval (Ventana Medical Systems) was selected for all immunostains: AMACR (P504S, ready to use, Biocare Medical, USA), β-catenin, (1:50, Cell Marque, USA), E-cadherin, (1:25, Life Technologies, Invitrogen, USA) and brachyury (1:200, Santa Cruz Biotechnology Inc., USA). Detection of AMACR, β-catenin and E-cadherin was performed with iView DAB Detection Kit (Ventana Medical Systems Inc., USA) and of brachyury with ultraView DAB Detection Kit (Ventana Medical Systems Inc., USA). Slides were counterstained with hematoxylin (Ventana Medical Systems Inc., USA) on an automated stainer and dehydrated in ethanol solutions (70%, 96%, and 100%) for one minute each. Before cover-slipping, sections were cleared in xylene for 2 minutes and mounted with Entellan (Surgipath, Germany).

Protein expression of germ layer markers was investigated by fluorescent immunohistochemical staining. Teratomas were fixed overnight in 4% paraformaldehyde and embedded in O.C.T solution for cryosectioning into 10 μm sections. For immunohistochemical analysis, cryosections were permeabilized with 0.5% Triton X-100 (Sigma) for 10 min and blocked with 2% BSA (Sigma) for 1 h at room temperature. Samples were incubated overnight at 4 °C with primary antibodies (1:100 dilution) against GATA4 (sc-25310, Santa Cruz Biotechnology, Santa Cruz, CA), BRACHYURY (sc-20109, Santa Cruz), and TUJ1 (sc-58888, Santa Cruz) representing endoderm, mesoderm, and ectoderm germ layers, respectively. Primary antibody-treated samples were then washed with PBS, stained with anti-rabbit Alexa Fluor 488 (A32731, Thermo Fisher Scientific) or anti-mouse Cy3-labeled IgG (072-01-18-06, KPL, Gaithersburg, MD, USA) secondary antibodies at 1:200 dilution for 2 h at room temperature, and counterstained with DAPI (1 mg/mL; Life Technologies). The stained samples were visualized by using confocal microscopy.

After resection of the tumors, they were fixed in neutralized 10% formalin solution and embedded in paraffin blocks using standard procedures. Paraffin sections were stained with hematoxylin-eosin (HE), and histopathological analysis was performed under a light microscopic observation. Tissue sections mounted on slides were also subjected to immunostaining after deparaffinization and rehydration, and antigen retrieval, according to the manufacturer’s protocol. Antibodies used in this study were anti-beta-III-tubulin (Abcam, ab264113, Cambridge, UK), anti-AFP (α-fetoprotein, Pierce, PA5-21004, Shreveport, LA, USA), anti-TRA-1-60 (Santa Cruz, sc-21705, Dallas, TX, USA), Brachyury (Santa Cruz, sc-374321, Dallas, TX, USA) and anti-PAR (BD Pharmingen, Franklin Lakes, NJ, USA). After several washes with PBS, bound antibodies were visualized using 3, 3′-diaminobenzidine according to the manufacturer’s protocol. The sections were counterstained with hematoxylin and mounted.

IOSE80 cells were cultured in TeSR
^TM maintenance media for 4 days. Subsequently, the STEMdiff
^TM kit (STEMCELL Technologies, Shanghai, China) was employed for one week, utilizing the Ectodermal Lineages, Mesodermal Lineages, and Endodermal Lineages kits sequentially. Afterwards, the differentiated Tri-lineage layer was selected and cultured in a dish with human amniotic epithelial cells (hAECs) as a feeder layer. The lineage layer was further cultured for 2 days to perform an immunofluorescence assay.
The lineage layer was fixed with 4% paraformaldehyde for 30 min and permeabilized for an additional 10 min with 0.1% Triton X-100 (Sigma-Aldrich, St Louis, USA). The blocking step was performed for 30 min using 5% FBS in PBS. The lineage layer was incubated with antibodies against Nestin (mouse anti-human; 1:200; Santa Cruz Biotechnology, Santa Cruz, USA), anti-Sox-17 (rabbit anti-human; 1:200; Santa Cruz Biotechnology), and brachyury (mouse anti-human; 1:200; Santa Cruz Biotechnology) for 1.5 h at 37°C. Each antibody was detected using corresponding secondary antibodies conjugated to fluorescein isothiocyanate.