Pbs t

Total protein was extracted from cells using RIPA buffer (Beyotime, Shanghai, China) supplemented with a protease inhibitor cocktail (BioDee, Beijing, China) (100:1) and phenylmethylsulfonyl fluoride (Beyotime, Shanghai, China) (100:1). Proteins were separated on 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels (GenScript, Nanjing, China). The proteins were transferred to polyvinylidene difluoride membranes, and then blocked with 5% non-fat milk for 1 h. The membranes were incubated with the primary antibodies CPT2 (1:1,000, NBP1-32226; Novus, Columbia, USA) and beta-actin (1:5,000, NB600-532SS; Novus, USA) for 1 h, and washed with PBST (Solarbio, China) three times, for 5 min each time. The membranes were then incubated with anti-rabbit secondary antibody conjugated with horseradish peroxidase (1:1,500, 7074; Cell Signaling Technology, Boston, MA, USA) for 1 h at room temperature, and washed three times using PBST. Protein bands were visualized using an enhanced chemiluminescence system (GE Healthcare, Fairfield, CT, USA).

Alkaline phosphatase staining was performed with the Vector Red Alkaline Phosphate Substrate Kit (Vector Laboratories, Burlingame, CA). For immunostaining, cells were fixed in 4% paraformaldehyde with 1% sucrose in phosphate buffered saline (PBS) (Invitrogen) for 15 min at room temperature. The cell membranes were then permeabilized with 0.5% TX-100 in PBS-T (Sigma-Aldrich), blocked in donkey serum (Sigma-Aldrich) and incubated with donkey serum containing primary antibodies including rabbit anti-OCT4 (Merck Millipore), rabbit anti-SOX2 (Abcam, San Francisco, CA) and rabbit anti-NANOG (Merck Millipore) at 1:100 dilutions, washed in PBS-T, and then incubated with Alexa Fluor 594 conjugated goat anti-rabbit secondary antibody (Cell Signaling Technology, Danvers, MA) at a 1:500 dilution. The cell nuclei were counterstained with DAPI. Fluorescent images were taken using a Zeiss or Nikon fluorescence microscope.

Total proteins extracted from harvested tissues and cells were prepared in mammalian lysis buffer containing phosphatase and protease inhibitors, and the protein concentration was determined using the BCA protein assay kit (Pierce Biotechnology, Rockford, IL, USA). The protein was fractionated on polyacrylamide-SDS gels and transferred to polyvinylidene difluoride membranes. The membrane was blocked with 5% nonfat milk in phosphate-buffered saline Tween 20 (PBS-T) and then incubated with a primary antibody against HO-1 (1 : 2000 v/v in PBS-T, Enzo Life Sciences, USA) or β-actin (1 : 2500 v/v in PBS-T, Cell Signaling Technology, MA) followed by incubation with a secondary antibody. Antibody binding was visualized with an ECL chemiluminescence system (GE Healthcare Bio-Sciences, Little Chalfont, UK).