Nanog

Cells were lysed in RIPA (radioimmunoprecipitation assay) buffer and the protein concentrations determined by the Bradford assay. Equal amounts of cell extracts were subjects to electrophoresis on sodium dodecyl sulphate–polyacrylamide gel and blotted onto nitrocellulose membrane (Millipore, Merck, Shanghai, China). After protein transfer, the membranes were blocked and then incubated with glyceraldehyde 3-phosphate dehydrogenase (Ambion, ThermoFisher Scientific), β-actin (Santa Cruz Biotechnology, Gene Company Ltd), p-AURKA (Thr288; Cell Signaling Technology, Gene Company Ltd), AURKA (Upstate, Gene Company Ltd), HA-tag (Sigma), c-Myc (Santa Cruz Biotechnology), SOX2 (Epitomics, Abcam, Hang Zhou, China) and Nanog (Epitomics, Abcam) primary antibodies at 4 ° overnight. The membranes were then incubated for 1 h at room temperature with the appropriate secondary antibodies. Proteins were detected with an enhanced chemiluminescence kit (Pierce, ThermoFisher Scientific).

Immunohistochemistry (IHC) was used to assess the protein expression of six CSC markers (CD133, CD44, ALDH1, SOX2, OCT4, and Nanog) and two EMT markers (E-cadherin and Snail-1). An automated immunostainer (Benchmark Ventana, Tucson, AZ) was used to stain tissue sections following the manufacturer's recommended procedure. The primary antibodies used against the follows: CD133 (1:200; Spring Bioscience, Pleasanton, CA), CD44 (1:200; Thermo Scientific, Fremont, CA), ALDH1 (1:100; BD Biosciences, San Diego, CA), SOX2 (1:100; Cell Signalling, Beverly, MA), OCT4 (1:100; Cell Marque, Rocklin, CA), Nanog (1:100; Epitomics, CA), E-cadherin (1:100; BD Biosciences, San Jose, CA) and Snail-1 (1:500; Santa Cruz Biotechnology, Santa Cruz, CA). IHC results were graded semiquantitatively based on the percentage of cells stained and the intensity of staining [34 (link)]. Briefly, the staining intensity was graded as weak (1+), moderate (2+), or strong (3+) and was multiplied by the percentage of positive cells. The total score was then classified as follows: 0-100 = grade 1, 101-200 = grade 2, and 201-300 = grade 3. Grade 2 or 3 tumors were considered to be positive for CSC markers and E-cadherin [35 (link)]. For Snail-1, tumors were considered positive when at least 10% of all tumor cells were immunoreactive [36 (link)].

Cells were seeded onto 12 mm coverslips in 24-well dishes and allowed for attachment. Cells were fixed in methanol, blocked in 3% BSA, and incubated with the following primary antibodies: Podoplanin (Santa Cruz, sc-376695), SPC1 (Santa Cruz, sc-13979), Ki67 (Abcam, ab15580), Nanog (Abcam, ab21624), β-catenin (Transduction Laboratories, #610154). After incubation with secondary antibody Alexa Fluor 488-conjugated goat anti-mouse (Jackson Immunoresearch, #115-545-003) or Alexa Fluor 594-conjugated goat anti-rabbit (Jackson Immunoresearch, #111-585-144), nuclei were stained with Hoechst 33342. Coverslips were mounted in Prolong Gold Antifade Reagent (Cell Signaling, #9071). Image acquisition was carried out using a Zeiss Axioplan2 microscope and the MediaView software.

The cells were plated on 20 mm coverslips and were fixed with 4% PFA for 20 minutes. Then, after washing with PBS three times for 10 minutes, the cells were successively treated with 0.2% Triton X‐100 (Sigma‐Aldrich) for 30 minutes, washed as above, and treated with 3% bovine serum albumin (BSA, Solarbio) at room temperature. Primary antibodies included OCT4 (1:100, Abcam), SSEA‐4 (1:100, Santa Cruz), NANOG (1:100, Abcam), TRA‐1‐60 (1:100, Santa Cruz), TNNT2 (1:100, Santa Cruz), α‐actinin (1:100, abcam), CX43 (1:100, Santa Cruz) and γ‐H2A.X (1:200, Abcam). Cells were washed and then incubated for 45 minutes at RT in the dark with 1:200 Alexa Fluor secondary antibodies (Life Technology). Cells were washed again as above, mounted with Fluoroshield Mounting Medium with DAPI (4, 6‐diamino‐2‐phenylindole), and imaged using a Confocal Microscope (Carl Zeiss, LSM 510 Meta).

Cells were grown on the surface of cover slides and fixed with 4% paraformaldehyde. After rehydration in PBS, the fixed cells were incubated with primary antibodies: γ-H2AX, β-catenin, active β-catenin and Nanog (Abcam) at room temperature for 1 h or at 4°C overnight. Alexa594-conjugated goat anti-mouse IgG (Invitrogen) or Alexa488-conjugated goat anti-rabbit IgG (Invitrogen) were used as secondary antibodies. The nuclei were stained with DAPI. Sections were examined by confocal microscopy (Olympus-FV1000, Tokyo, Japan).

Cells were were fixed in 4% paraformaldehyde (PFA) for 15 min, permeabilized with 0.1% Triton X-100 for 30 min, and then incubated with blocking solution (PBS + 1% BSA) at 4°C for 30 min followed by primary antibodies at 4°C overnight. Primary antibodies were diluted in Immnol Fluorence Staining Primary Antibody Dilution Solution (Beyotime, P0108) at the followings ratios: OCT4 (rabbit polyclonal antibody, 1:500, Abcam), SOX2 (rabbit polyclonal antibody, 1:500, Abcam), NANOG (rabbit polyclonal antibody, 1:500, Abcam), SSEA4 (rabbit polyclonal antibody, 1:500, Abcam), CD105 (rabbit monoclonal antibody, 1:500, Abcam), β-Ⅲ-tubulin (rabbit monoclonal antibody, 1:500, Chemicon), AFP (mouse monoclonal antibody, 1:500, Chemicon), Desmin (mouse polyclonal antibody, 1:500, Abcam), Nestin (rabbit monoclonal antibody, 1:200, Chemicon), PDX1 (rabbit monoclonal antibody, 1:500, Chemicon) and α-actin (rabbit monoclonal antibody, 1:200, Sigma). After washing three times with PBS, cells were incubated with Alexa Fluor 488 (donkey anti-mouse) or Alexa Fluor 546 (goat anti-rabbit) conjugated secondary antibody (1:500; Chemicon) for 1h at room temperature in the dark. Finally, DNA was stained with Hoechst33342 (Beyotime, C1005) for 3 min. Negative controls were processed the same way, except that the primary antibodies were replaced with blocking buffer.