Oct3 4

For immunostaining, cells were seeded onto eight-well Nunc Lab-Tek II Chambered Coverglass (ThermoFisher Scientific, Waltham, MA, USA, #155411), fixed, and permeabilized with 4% PFA (paraformaldehyde) in DPBS (Dulbecco’s modified PBS) for 15 min on RT (room temperature). Next, cells were blocked for 60 min at RT in a blocking solution (DPBS supplemented with 2 mg/mL BSA, 0.1% Triton-X 100, and 1% fish gelatin with or without 5% goat serum). Then, the samples were incubated for 60 min at RT in the blocking solution supplemented with the following primary antibodies: OCT3-4 (1:50, Santa Cruz, CA, USA, #SC-5279) and NANOG (1:100, R&D Systems, Minneapolis, MN, USA, #AF1997) as pluripotency markers; AFP (1:500, Sigma, St. Louis, MO, USA#A8452), SMA (1:500, Abcam, Cambridge, UK, #ab7817), and ß-III-tubulin (1:2000, R&D Systems, Minneapolis, MN, USA, #RD-MAB1195) as markers specific for the three lineages. After washing with DPBS, the cells were incubated for 60 min at RT with Alexa Fluor 647-conjugated goat anti-mouse and Alexa Fluor 594-conjugated donkey anti-goat IgG antibodies (1:250, ThermoFisher Scientific, Waltham, MA, USA, #A11029, #A11012). DAPI (ThermoFisher Scientific, Waltham, MA, USA, # D1306) was used for nuclear staining. The samples were then examined by a Zeiss LSM 710 confocal laser scanning microscope.

The western blot experiment was performed as described previously 20 and the antibodies used were: IFITM1/2/3 (F12; sc‐374026; Santa Cruz Biotechnology), NANOG (sc‐293121; Santa Cruz Biotechnology), OCT3/4 (SC‐5279; Santa Cruz Biotechnology), SOX2 (AB5603, Millipore, Billerica, MA, USA), β‐actin (sc1616R; Santa Cruz Biotechnology) and IFITM3 (AF3377; R&D Systems). The protein bands were detected by Amersham ECL Prime western blot detection reagent (RPN2232; GE Healthcare, Chicago, IL, USA).

Cultured cells were fixed with 4% paraformaldehyde for 20 min at room temperature, made permeable with 0.4% Triton X-100 in PBS for 15 min and blocked with 3% BSA in PBS for 1 hour. Cells were incubated overnight at 4°C with primary antibodies diluted in 1% BSA in PBS. Primary antibodies used were Oct3/4 (Santa Cruz; rabbit, 1:500), Sox17 (R&D Systems; goat, 1:250), FoxA2 (Novus Biologicals; Mouse, 1:1000), HNF4a (Santa Cruz; goat, 1:250), AFP (Sigma; mouse, 1:1000), and Albumin (DAKO; rabbit, 1:500). Primary antibodies were probed with respective secondary antibodies conjugated to Alexa Fluor 488 or 594 (Molecular Probes; 1:1000) and nuclei were visualized with DAPI.

Cells were washed twice in ice-cold PBS, and lysed in JS buffer (50 mM HEPES pH 7.5 containing 150 mM NaCl, 1% Glycerol, 1% Triton X100, 1.5 mM MgCl₂, 5 mM EGTA, 1 mM Na₃VO₄, and 1X protease inhibitor cocktail). Protein concentration was determined by the Bradford assay (BioRad, Milan, Italy) using bovine serum albumin as the standard, and equal amounts of proteins were analyzed by SDS-PAGE (12.5% acrylamide). Gels were electroblotted onto nitrocellulose membranes (G&E Healthcare, Milan, Italy). Membranes were blocked for 1 h with 5% non-fat dry milk in Tris Buffered Saline (TBS) containing 0.1% Tween-20, and incubated at 4°C overnight with the primary antibody. Detection was performed with peroxidase-conjugated secondary antibodies using an enhanced chemiluminescence system (ThermoEuroclone, Milan, Italy). Primary antibodies used were: anti-Zeb-1, -Oct 3/4, -Nanog, -cytokeratin 18, and -cytokeratin 8 (Santa Cruz Biotechnologies, MA, USA), anti-DNMT3b (Abcam, MA, USA), and anti-β-actin (Sigma Aldrich, Milan, Italy).

Cells were fixed in 4% PFA diluted in PBS and kept overnight at 4°C. The samples were quenched with 50 mM NH₄Cl in PBS for 10 min at room temperature and permeabilized with 0.3% Triton X-100 and 5% FBS in PBS for 30 min. Antibodies were diluted in PBS containing 5% FBS and incubated for 1 h at room temperature. Between antibodies, cells were washed three times in PBS. Nuclei were stained for 10 min with DAPI (D1306; Thermo Fisher Scientific) diluted 1:10,000 in PBS. Coverslips were mounted on glass slides with DAKO mounting medium (DAKO, S3023). Antibodies used were CAT (D4P7B) XP (12980, 1:700; Cell Signaling), PEX14 (ab183885, 1:400; Abcam), PMP70 (ab211533, 1:400; Abcam), Tom20 (#11802-1-AP, 1:100; Invitrogen), OCT3/4 (SC5279, 1:100; Santa Cruz), TRA-1-60 (#MAB4360, 1:100; Millipore), TRA-1-81 (#MAB4381. 1:100; Millipore), anti-rabbit-Alexa Fluor 488 (A11034, 1:500; Life Technologies), Donkey anti-mouse IgG-594 (A21203, 1:300; Thermo Fisher Scientific), and anti-mouse Alexa Fluor 546 (A11003, 1:500; Life Technologies).