Anti oct4a

Intestinal tissue and cells were lysed in RIPA lysis buffer (Beyotime, P0013B) with protease inhibitor cocktail (Sigma-Aldrich, P8340), phosphorylase inhibitor (Millipore, 361515) and PMSF (R&D Systems, 4486/50). Then the 4× loading buffer (which is free of SDS for CD47) was added into the cell lysate. The mixture was heated on 60–65 °C for 5 minutes for CD47 while others were heated on 95–99 °C for 8 minutes. Protein levels were analyzed via Western blot using selective antibodies and normalized against the level of GAPDH. The following antibodies were used: anti-c-Myc (Cell Signaling, 2840 s), anti-KLF4 (Cell Signaling, 4083 s), anti-Oct-4A (Cell Signaling, 2840 s), anti-Sox2 (Cell Signaling, 23064 s), anti-CD47 (R&D, BAF1866), anti-GAPDH (Santa Cruz Biotechnology, sc-32233). Quantification of bands was analyzed by software Image J software.

Cells were seeded in four-well plates. Cells were fixed in 4% paraformaldehyde for 15 min, incubated in 0.1 M glycine for 10 min at room temperature (RT), and then in blocking solution composed of 5% goat serum, 0.6% Triton in PBS for 30 min at RT. Cells were immunostained at 4°C overnight in blocking solution with primary antibodies anti-Sox2 (1:300; Millipore), anti-Oct4A (1:400; Cell Signaling), anti-Nanog (1:500; Abcam), anti-SSEA4 (1:50; Millipore), and anti-TRA-1–81 (1:50; Millipore). For the immunofluorescence characterization, samples were incubated with appropriate secondary antibodies Rhodamine-Red anti-mouse IgM and antirabbit IgG, Alexa Fluor 488 anti-mouse IgG, (Jackson ImmunoResearch, distributed by Li StarFish, Milan, Italy) for 1 hr at RT, and nuclei were counterstained with Hoechst 33258. Cells were mounted with GelMount aqueous mounting media (Sigma). The images were acquired using a Leica DMI4000B inverted microscope linked to a DFC360FX or a DFC280 camera (Leica Microsystems).

The iPSCs or embryoid bodies that spontaneously differentiated were grown in a suitable medium on glass coverslips placed in a 12-well plate. The grown cells were washed with 1× PBS; fixed and permeabilized in chilled methanol (Samchun Chemicals, Korea) for 10 min; washed with PBS; and blocked with a 3% BSA solution in PBS (Thermo Fisher Scientific, Inc.) for 30 min. After that, the cells were incubated with a primary antibody at 4°C overnight. The next day, the cells were rigorously washed and incubated with Alexa Fluor 488– or Alexa Fluor 546–conjugated secondary antibodies (Invitrogen, Carlsbad, CA, USA) for 1 h at room temperature. After a wash with PBS, nuclei were stained with a Hoechst 33258 solution (5 μM; Sigma-Aldrich) for 10 min. For staining of pluripotency markers of iPSCs, the following primary antibodies were used: anti-TRA1-60 (1 : 500; Abcam), anti-SSEA4 (1 : 500; Abcam), anti-OCT4A (1 : 1000; Cell Signaling Technology), and anti-NANOG (1 : 1000; Cell Signaling Technology). To detect germ layer markers of differentiation, the following primary antibodies were used: anti-AFP (for endoderm; 1 : 100; Santa Cruz Biotechnology), anti-SMA (for mesoderm; 1 : 250, Sigma), and anti-TUJ-1 (for ectoderm; 1 : 250; Abcam). All images were captured using the fluorescence microscope (Olympus IX53; Olympus Corporation, Tokyo, Japan).

Anti-CD45, anti-EpCAM, anti-fibronectin, anti-ALDH1A1, anti-CD133, anti-vimentin, and anti-cytokeratin antibodies were from Abcam. Anti-snail, anti-Oct-4A, and anti-CD44 antibodies were from Cell Signaling. Anti-E-cadherin, anti-N-cadherin, anti-CD24, anti-CD44, Anti-CD45, anti-cytokeratin, and anti-tubulin antibodies were from BD Transduction Laboratories.