Nanog

ICC analyses were performed to evaluate the expression of genes related to pluripotency and differentiation. Before staining, all cell samples were preincubated for 10 min at 4°C and fixed with 4% paraformaldehyde for 30 min. After washing twice with Dulbecco’s phosphate-buffered saline (DPBS; Welgene), samples were treated for 1 h with 10% goat serum in DPBS to prevent nonspecific binding. Serum-treated cells were incubated overnight at 4°C with primary antibodies. The primary antibodies used were as follows: OCT4 (Santa Cruz Biotechnology, CA, USA; 1:200), SOX2 (Millipore; 1:200), NANOG (Santa Cruz Biotechnology; 1:200), SSEA1 (Millipore; 1:200), SSEA4 (Millipore; 1:200), Neurofilament (Millipore; 1:100), Vimentin (Millipore; 1:100), and Cytokeratin 17 (Millipore; 1:100). When we used the antibodies for intracellular proteins such as OCT4, SOX2, and NANOG, fixed cells were treated for 5 min with 0.2% Triton-X100 (Sigma-Aldrich, MO, USA) before serum blocking. After incubation with the primary antibody, the cells were treated for 3 h at room temperature with Alexa Fluor-conjugated secondary antibodies. Nuclei were stained with Hoescht 33342 (Molecular Probes). Images of stained cells were captured using a LSM 700 Laser Scanning Microscope (Carl Zeiss, Germany) and processed with the ZEN 2012 Light Edition program (Carl Zeiss).

mRNA expression analyses were carried out by real-time PCR using a DNA Opticon system (MJ Research, Waltham, MA) and using DyNAmo SYBR green qPCR mix (New England Biolabs, Ipswich, MA). Standard curves were generated by serially diluting the sample expected to have the most amount of the PCR product. The yield of product for each unknown sample was calculated by applying its threshold cycle, or C(T), value to the standard curve using the Opticon Monitor analysis software (version 1.01, MJ Research, Waltham, MA). Values were normalized to corresponding 18S rRNA values and expressed as the fold increase relative to controls. Primers for HER2, HIF-1α, BCRP, GAPDH, BMI-1, Nanog and TWIST were obtained from (Sigma, St. Louis, MO or Qiagen Valencia, CA).

Before fixation, cell samples were preincubated for 10 min at 4°C and fixed with 4% paraformaldehyde for 30 min. After washing twice with DPBS (Welgene), samples were treated for 1 h with 10% goat serum in DPBS to prevent nonspecific binding. Serum-treated cells were incubated overnight at 4°C with primary antibodies. The primary antibodies used were as follows: OCT4 (Santa Cruz Biotechnology; 1 : 200), SOX2 (Millipore; 1 : 200), NANOG (PeproTech; 1 : 200), SSEA1 (Millipore; 1 : 200), and SSEA4 (Millipore; 1 : 200). When antibodies against intracellular proteins such as OCT4, SOX2, and NANOG were used, fixed cells were treated for 15 min with 0.1% Triton-X100 (Sigma–Aldrich) before serum blocking. After incubation with the primary antibody, the cells were treated for 3 h at room temperature with Alexa Fluor-conjugated secondary antibodies. Nuclei were stained with Hoechst 33342 (Molecular Probes). Images of stained cells were captured using an LSM 700 laser scanning microscope (Carl Zeiss) and processed with the ZEN 2012 Light Edition program (Carl Zeiss).