Bb 3209

The total protein was extracted by using radio-immunoprecipitation assay (RIPA) lysis buffer (BB-3209, BestBio Science, Shanghai, China). The proteins were separated using sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), and transferred onto a polyvinylidene fluoride (PVDF) membrane with constant voltage of 80 V. After the membranes were blocked for 1 h, they were incubated with diluted primary antibodies, rabbit anti-human PTEN (1: 100, ab32199; Abcam Inc.. Cambridge, MA, USA), vascular endothelial growth factor (VEGF) (1: 100, sc4570, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), matrix metalloproteinase (MMP)-2 (1: 100, ab37150, Abcam Inc.. Cambridge, MA, USA), MMP-9 (1: 100, ab73734, Abcam Inc.. Cambridge, MA, USA), protein kinase B (Akt) (1: 100, ab8805, Abcam Inc.. Cambridge, MA, USA), p-Akt (1: 100, ab192623, Abcam Inc.. Cambridge, MA, USA), and GAPDH (1: 100, ab9385, Abcam Inc.. Cambridge, MA, USA) (internal reference) for 1 h at 37 °C. TBST washes (5 min × 3) were followed. Horseradish peroxidase (HRP)-labeled rabbit anti-human IgG (1: 20000, ab205718, Abcam Inc.. Cambridge, MA, USA) served as the secondary antibody. The membrane was developed by enhanced chemiluminescence (ECL).

Total protein was extracted by using radioimmunoprecipitation assay cell lysis buffer (BB-3209, BestBio Inc., Shanghai, China). Extracted protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride membrane under constant voltage conditions. The membrane was blocked with sealing solution for 1 h and was then incubated overnight at 4 °C with diluted rabbit polyclonal antibodies against HOXA11 (1:500, ab72591), CD133 (1:500, ab19898), CD44 (1:2000, ab157107), Nanog (1:1000, ab106465), SOX2 (1:1000, ab97959), and Oct4 (1:1000, ab137427) and with rabbit monoclonal antibodies against β-catenin (1:5000, ab32572), NKD1 (1:10000, ab133650), c-myc (1:1000, ab32072), and cyclinD1 (1:10,000, ab134175). On the following day, the membrane was further incubated with horseradish peroxidase-labeled secondary antibody, goat anti-rabbit immunoglobulin G (IgG) (1:2000, ab205718), at 37 °C for 1 h. All antibodies were purchased from Abcam (Cambridge, MA, USA). Finally, the membrane was washed three times with PBS (5 min/wash) and developed. The relative expression levels of the target proteins were calculated as the ratio of the gray value of the target protein band to that of the internal reference band (GAPDH). All experiments were repeated three times.

The tissue and cellular proteins were isolated using radioimmunoprecipitation assay lysis buffer (BB-3209, BestBio, Shanghai, China). The supernatant was collected after high-speed centrifugation and the protein concentration was determined using a bicinchoninic acid assay kit (Thermo Fisher Scientific). After protein separation by sodium dodecyl sulfate polyacrylamide gel electrophoresis, the proteins were transferred onto a polyvinylidene fluoride membrane at a constant voltage of 80 V. After blocked for 1 h, the membrane was incubated overnight at 4 °C with primary antibodies: rabbit anti-human NRMT (ab72660, 1:500, Abcam, Cambridge, UK), CENPA (ab13939, 1:1000, Abcam), Myc (ab32, 1:1000, Abcam), Bcl2 (ab185002, 1:1000, Abcam), poly-ADP-ribose polymerase (PARP) (556494, 1:1000, BD-Pharmingen, San Diego, CA), caspase 3 (ab32351, 1:1000, Abcam), and Cleaved caspase 3 (ab32042, 1:1000, Abcam). The goat anti-rabbit immunoglobulin (IgG) (ab205718, 1:10,000, Abcam) as the secondary antibody was added for 1-h incubation at 37 °C. The membrane was developed by enhanced chemiluminescence and photographed by SmartView Pro 2000 (UVCI-2100, Major Science, Saratoga, CA). The band intensity was quantified using Quantity One software. Each set of experiments was repeated three times.

Radio-immunoprecipitation assay cell lysis buffer (BB-3209, BestBio, Shanghai, China) was employed to lyse and extract the total protein from the cells. The extracted proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto a polyvinylidene fluoride membrane at a constant voltage of 80 V. Then, the membrane was blocked for 1 h and was incubated at 37 °C for 1 h with the following primary antibodies: rabbit polyclonal antibodies directed against FOXA1 (ab23738, 1:1000), Bcl-2-associated X protein (Bax, ab199677, 1:1000), B cell lymphoma 2 (Bcl-2, ab196495, 1:500), and cleaved caspase-3 (ab49822, 1:1000). All of these antibodies were purchased from Abcam (Cambridge, MA, USA). The membrane was then washed three times with phosphate-buffered saline (PBS) at room temperature (5 min each time), followed by coloration. The relative expression of the target protein was equal to the gray value of the target protein band divided by the gray value of the internal reference band (the internal reference was glyceraldehyde-3-phosphate dehydrogenase (GAPDH)).