Anti pcna

Immunofluorescence was used to detect the expression of L-PGDS in tumor tissue (1 : 50; Bioworld, Louis Park, MN). The secondary antibodies were Alexa Fluor 555-labeled donkey anti-rabbit IgG (1 : 300, Invitrogen, Carlsbad, CA). Images were acquired using microscopy (Nikon, Tokyo, Japan). For immunohistochemical analysis, the tumor tissues were fixed in 4% paraformaldehyde (PFA), embedded in paraffin, and cut into 5 μm thick sections. Then, these sections were incubated with anti-PCNA (1 : 100; Bioworld, Louis Park, MN) overnight at 4°C and, subsequently, incubated with the secondary antibody at 37°C for 30 min. Finally, tissues were counterstained with 3,3′-diaminobenzidine (DAB) and photographed by microscopy.

The antibodies specifically against SOX2, c-MYC, 14-3-3, and β-Catenin were purchased from Abcam (UK); anti-CD44 and anti-KLF4 were products of Proteintech (USA); anti-β-actin, HRP-conjugated anti-mouse or anti-rabbit IgG were purchased from ZSGBBIO (CHN); anti-CBY1 was purchased from Santa Cruz (USA); anti-SP3 was purchased from Sigma-Aldrich (USA); anti-Nanog, anti-phosphorylated-β-Catenin, anti-GAPDH and anti-PCNA were obtained from Bioworld (USA); and FITC-labeled goat anti-rabbit IgG was from Sigma-Aldrich (USA). BSA was a product of Sigma-Aldrich (USA); bFGF was obtained from Proteintech (USA); EGF and B27 supplement were from Invitrogen (Thermo Fisher, USA), and ultra-low attachment plates were purchased from Corning Corp. (USA).

Cells were lysed and homogenized in RIPA buffer supplemented with complete protease inhibitors. Equal amount of proteins (200 µg) was resolved in 12% SDS-PAGE. The proteins were then transferred onto PVDF membranes following electrophoresis. After blocked in 5% (w/v) non-fat milk for 1 h at room temperature, the membranes were then incubated at their respective appropriate dilutions of specific primary antibodies overnight at 4°C. The sources of primary antibodies were: anti-Oct4, anti-Sox2, anti-Vimentin and anti-phospho-STAT3 (Signalway Antibody, USA), anti-GAPDH (Kangcheng, Shanghai, China), anti-E-cadherin, anti-ERK1/2, anti- phospho-ERK1/2 and anti-N-cadherin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-PCNA, anti-STAT3, anti-NF-κB, anti-phospho-NF-κB, anti-CyclinD1, anti-VEGF and anti-C-Jun (Bioworld Technology, Louis Park, MN, USA), anti-C-myc (ProteinTech Group, Chicago, IL, USA).