Cd133

For protein extraction, all samples were treated with 200 μL of lysis buffer. Following lysing, 50 μg of protein from each sample was loaded into the wells of a sodium dodecyl sulfate-polyacrylamide gel and subjected to electrophoresis at 50 V for a duration of 4 h. Subsequent to electrophoresis, the proteins were transferred onto poly(vinylidene fluoride) membranes. After a 1 h incubation in a blocking buffer, the membranes were exposed to primary antibodies. These primary antibodies included CD133 (1:200; Sigma; ZRB1013; USA), OCT4 (1:500; Abcam; Cambridge, UK), SOX2 (1:500; Abcam; UK), and β-actin (A5441; 1:20,000; Sigma; USA). Incubation with these primary antibodies was carried out at 4 °C for 16 h. Subsequently, the membranes were incubated with secondary antibodies for 90 min. The secondary antibodies employed were goat anti-rabbit (AP132P, 1:5000; Millipore, Billerica, MA, USA) and goat anti-mouse antibodies (AP124P, 1:5000; Millipore). Specific protein bands were visualized using an enhanced chemiluminescence solution (Western Lightning, 205-14621; Perkin Elmer, Waltham, MA, USA), and image acquisition and analysis were performed using the MiniChemiTM imaging and analysis system (Beijing Sage Creation, Beijing, China).

The CD133 (GenBank: NM_006017) gene was purchased from the National RNAi Core Facility (Taipei, Taiwan) and used in construction of the pLAS5w.Pbsd recombinant lentivirus-expressing vector (Invitrogen, Waltham, MA, USA). SKOV3 cells at a density of 10⁵ cells/well were seeded in 24-well plates. After overnight incubation, 2 mL of fresh media containing 8 μg/mL polybrene (Sigma-Aldrich) and the recombinant lentivirus were added. Following transduction and puromycin (Sigma-Aldrich) selection, SKOV3 cells with stable CD133 overexpression were used for subsequent assays.

Western blot assay was performed referring to several previously published works [24–26 (link)]. Briefly, proteins were extracted using Radio Immunoprecipitation Assay (RIPA) lysis buffer (Beyotime, Shanghai, China) and separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, Bio-Rad, Hercules, CA, USA). The separated proteins were subsequently electro-transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA), which were then soaked in 5% nonfat milk for a while. The membranes were then incubated with primary antibodies against ZNF267 (HPA003866, Sigma-Aldrich, Darmstadt, Germany), E-cadherin (SAB4503751), N-cadherin (SAB5700641), snail (SAB1306281), CD44 (SAB4300691), CD133 (SAB4300882), OCT4 (SAB5100006), and GAPDH (G9545, Sigma-Aldrich), followed by another one hour of incubation with secondaries (A0545, 1:10,000, Sigma-Aldrich). Finally, the blotting was enhanced using ECL reagents (Pierce, Rockford, IL, USA).

Adherent cells on passage 7 were resuspended in FACS (fluorescence-activated cell sorting) buffer, and the concentration adjusted to 10⁵cells/mL. For intracytoplasmic and nuclear markers, cells were permeabilized with 5 μl 0.1 % Triton X-100 for 30 min prior to incubation with primary antibodies (concentration of 1:100) specific for stem cells, inflammation, and cell cycle progression: Oct 3/4 (C-10, SC-5279), Nanog (n-17, SC-30331), CD45/OX1 (SC-53045), CD105 (2Q1707, SC-71042), CD90 (Thy-1, aTHy-1A1), CD34 (BI-3C5, SC: 19621), Caspase-3 (SC-7272), HSP-47 (SC-8352), P21 (SC-6246), Ki67 (Ab – 15580), Cyclin-D1 (AB-27618), P53 (Ab −26), TRA-1-81 (SC-21706), MCP-1 (SC- 32771), TNF-R1 (SC, 52746), all from Santa Cruz Biotechnology, as well as CD117 (c-Kit, SCF-Receptor Ab-6, RB-1518-R7, Thermo Scientific, Lab Vision Corporation, Fremont, CA, USA), VEGF-R1 (Clone VG1, M7273, DakoCytomation, CA, USA), COX-2 (Cayman Chemical Co, EUA), CD11b (MCA-551FT), Ly6a (Ab – 51317), CD1a (SC-18885), and CD133 (Mab4310, Merck Millipore), all for 45 min at room temperature. Then, cells were incubated for 2 h with a secondary antibody (Anti-Mouse FITC, DakoCytomation, Santa Cruz Biotechnology). The analysis was performed using a flow cytometer (FACSCalibur, BD). The expression of surface markers was determined by comparison with an isotype control analyzed by a histogram on the CELLQUEST program.

Western blotting was performed according to a standard protocol, as described previously [16 (link)]. The total proteins were collected using SDS lysis buffer (Beyotime, Shanghai, China, P0013G), and protein concentrations were determined by Bicinchoninic Acid (KeyGen, Nanjing, China, KGP902). Nuclear extracts were obtained using the NE‐PER Nuclear and cytoplasmic extraction reagents (Thermo Scientific, Waltham, MA, USA, 78833) according to the manufacturer’s instructions. The following primary antibodies were used: FUBP1(ABE1330) from Merck Millipore (Boston, MA, USA); CD133 (#86781), ALDH1 (#54135), CD44 (#37259), p‐GSK3β (Ser9) (#9323), GSK3β (#9832), n‐p‐β‐catenin (Ser33/37/Thr41)(#8814), β‐catenin (#9582), Histone (4499), and c‐Myc (#13987) from Cell Signaling Technology (Boston, MA, USA); LGR5 (ab75732) and DVL1 (ab233003) from Abcam (Cambridge, UK); and β‐actin (A5441) from Sigma‐Aldrich (St. Louis, USA). HRP‐conjugated anti‐rabbit IgG (Cell Signaling Tech, #7074) and anti‐mouse IgG (Sigma‐Aldrich, AP308P) were used as secondary antibodies. Proteins were determined using ECL Plus Reagent (Millipore, WBKLS0100).