Klf4 d1f2

Cell pellets were lysed with SDS Lysis Buffer (1% SDS, 10 mM Tris pH 7.4, 1 mM phenylmethylsulfonyl fluoride) and supplemented with Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher). Protein samples were resolved by SDS-PAGE (Novex NuPage Bis Tris Gel; Invitrogen MiniGel Tank) and then transferred onto PVDF membranes (iBlot 2 system, Invitrogen). The following antibodies were used: KLF4 D1F2 from Cell Signaling, anti-Actin A5316 from Sigma, Direct-Blot™ HRP anti-β-actin Antibody, GAPDH Loading Control Monoclonal Antibody (GA1R), PARP antibody from Cell Signaling, and MYC antibody from Cell Signaling. Primary antibodies were used at 1:1,000 dilution. Primary actin antibody was used at 1:100,000 dilution, or HRP anti-β-actin at 1:300,000. HRP cross-linked secondary antibodies (anti-rabbit IgG, HRP-linked Antibody #7074 and anti-mouse IgG, HRP-linked Antibody #7076 from Cell Signaling) were detected by West Femto Maximum Sensitivity Substrate (Thermo Fisher) and Amersham Hyperfilm ECL (GE). All secondary antibodies are used at 1:15,000-1:30,000 dilution. Protein quantity was normalized based on housekeeping control bands.

Human liver cancer cell lines, HepG2, PLC, and HepB3 were obtained from American Type Culture Collection (Manassas, VA, United States). Huh7 was obtained from the Japan Society for the Promotion of Science (Tokyo, Japan). These cells were maintained in 1,640 medium (Gibco, Thermo Fisher, Waltham, MA, United States) supplemented with 10% heat-inactivated fetal bovine serum (Hyclone) and penicillin/streptomycin at 37°C in a humidified atmosphere of 5% CO2.
The reagents used in this study were: Antibodies of anti-GINS1 (ab181112), anti-BMI1 (ab126783), and anti-RAS (ab52939) were purchased from Abcam (Cambridge, United Kingdom). KLF4 (D1F2) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; D16H11) were obtained from Cell Signaling Technology (Danvers, MA, United States).

OSCC cells were lysed in lysis buffer containing protease and phosphatase inhibitors. Protein samples of 20–30 µg were loaded into sodium dodecyl sulphate–polyacrylamide gel and separated by electrophoresis. After the proteins were transferred onto polyvinylidene difluoride (PVDF) membrane, the membrane was probed with primary antibodies, followed by second antibodies. The protein signals were developed after incubation with chemiluminescent substrates. Antibodies against KLF4 (D1F2) (12173S, 1:1000 dilution), Nanog (D2A3) (8822S, 1:1000 dilution), Sox2 (D6D9) (3579S, 1:1000 dilution), and β‐actin (3700S, 1:5000 dilution) were purchased from Cell Signaling Technology (MA, USA). Anti‐FOXA2 [EPR4466] (ab108422, 1:1000 dilution), and anti‐FTO antibody [EPR6894] (ab126605, 1:1000 dilution) were from Abcam (Cambridge, UK).