Cell culture and reagents
The p53−/−MEFs, HRASV12-expressing p53−/−MEFs, TIG-3, and TIG-3–SMR cells were prepared as previously described13,14. These cells were cultured in DMEM (Nissui, Japan) containing 10% foetal bovine serum (FBS). HCT116, SW480, and DLD1 colon cancer cell lines and HCC827 and H460 lung cancer cell lines were purchased from ATCC (Manassas, VA, USA). HCT116 cells were cultured in McCoy's 5A medium (Gibco, Waltham, MA, USA), SW480 and DLD1 cells were grown in DMEM, and HCC827 and H460 cells were cultured in RPMI 1640 medium (Nissui, Japan), each containing 10% FBS. All cells were tested for mycoplasma contamination using the MycoAlert™ Mycoplasma Detection Kit (Lonza, Switzerland), which confirmed the absence of mycoplasma. U0126 (Promega, Madison, WI, USA), LY294002 (Fujifilm Wako, Japan), PD184352, dinaciclib, palbociclib (Selleck Biotech, Houston, TX, USA), thiamet G (Sigma-Aldrich, St. Louis, MO, USA), and OSMI1 (Cayman Chemical, Ann Arbor, MI, USA) were used in the inhibition experiments. MG-132, and anti-c-H-Ras (OP23) and anti-SV40 T antigen (DP01) antibodies were obtained from Calbiochem (Merck, Germany). Anti-OGT (H-300), anti-TERT (H-231), and anti-c-Myc (9E10) antibodies were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-O-GlcNAc antibody (CTD110.6) was purchased from Covance (Princeton, NJ, USA). Anti-SOX2 (D9B8N), anti-ERK1/2 (#9102), and anti-pERK1/2T202/Y204 (#9101) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-CDK1 (A2861), anti-pCDK1T161 (AP0324), and anti-CDK2 (A0294) antibodies were obtained from ABclonal (Cambridge, MA, USA). Anti-β-actin (AC-74) was obtained from Sigma-Aldrich. All antibodies were used at a 1:1000 dilution.
Expression vectors and virus infection
A SOX2 sgRNA CRISPR/Cas9 All-in-One Lentivirus vector set (Applied Biological Materials, Canada) was used for knockout of the SOX2 gene. The sgRNA sequences were as follows: sgRNA#1, CAACCAGAAGAACAGCC; sgRNA#2, TCATCGACGAGGCCAAG; and sgRNA#3, ATTATAAATACCGGCCG. For knockout of the p53 gene in TIG-3 cells, the lentivirus vector pSpCas9(BB)-2A-Puro (PX459) V2.0 was obtained from the Addgene repository (plasmid #62988, Cambridge, MA, USA), and the target sequence for p53 (ACCAGCAGCTCCTACACCGG) was cloned into the PX459 vector following the repository’s recommended Zhang lab protocol. Retroviral vectors pMXs-mSOX2-IP (plasmid #15919), pBabe BRAFV600E (plasmid #17544), and pBabe PI3K p110CAAX (plasmid #13339) containing the puromycin resistance marker were purchased from Addgene. The pBabe SV40 T antigen, pBabe c-Myc, and pBabe HRASV12 vectors were used with blasticidin, neomycin, and hygromycin selectable markers, respectively. Lentiviral and retroviral infections were performed as previously described23,64. The infected cells were selected with the appropriate antibiotics.
siRNA and transfection
The predesigned short interfering RNA (siRNA) for mouse Cdk1 and Cdk2 were obtained from Sigma-Aldrich: siCDK1#1, SASI_Mm01_00179321; siCDK1#2, SASI_Mm01_00179322; siCDK2#1, SASI_Mm02_00323492; siCDK2#2, SASI_Mm01_00151932. We performed reverse transfection of siRNA using Lipofectamine™ RNAiMAX Transfection Reagent (Thermo Fisher Scientific, Waltham, MA, USA).
Cells were lysed with TNE buffer (10 mM Tris-HCl, pH 7.4; 1% NP-40; 150 mM NaCl; 1 mM EDTA; 1 mM DTT, and protease inhibitor cocktail) (Nacalai Tesque, Japan). Protein concentrations were measured using the Bradford assay. Cellular proteins (20 µg) were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to polyvinylidene difluoride membranes (Merck). The membranes were probed with primary antibodies, followed by incubation with horseradish peroxidase-conjugated mouse or rabbit immunoglobulin G (GE Healthcare, England) and visualisation using Chemi-Lumi-One Super or Ultra assay kits (Nacalai Tesque). The protein bands were digitalised using the LAS-3000 mini image analyser (Fujifilm, Japan).
Quantitative real-time PCR
Total RNA was extracted using the NucleoSpin RNA kit (Macherey-Nagel, Germany) following the manufacturer’s instructions. Double-stranded cDNA was prepared from total RNA using oligonucleotide (dT), random primers, and Superscript III reverse transcriptase (Invitrogen, Carlsbad, CA, USA). Quantitative real-time PCR (qPCR) analysis was performed as previously described23. The following probes were predesigned from Applied Biosystems® (Thermo Fisher Scientific): mouse β-actin, Mm00607939_s1; mouse Oct4, Mm03053917_g1; mouse Klf4, Mm00516104_m1; mouse Sox2, Mm03053810_s1; human β-actin, Hs00357333_g1; human OCT4, Hs03005111_m1; human KLF4, Hs00358836_m1; human SOX2, Hs01053049_s1; and human NANOG, Hs04260366_m1.
The p53−/− MEFs and HRASV12-expressing p53−/− MEFs (4 × 104) were cultured on glass coverslips in 6-well plates and treated with U0126 (10 µM) or LY294002 (20 µM) for 24 h. The cells were washed in phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde in PBS for 15 min, and permeabilised with 0.5% Triton X-100 in PBS for 10 min. Permeabilised cells were blocked with Blocking One Histo reagent (Nacalai Tesque) for 30 min and incubated with anti-SOX2 antibody for 1 h at room temperature. Alexa Flour 488-conjugated anti-rabbit antibody (Thermo Fisher Scientific) was used as the secondary antibody for 1h at room temperature. VECTASHIELD Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA, USA) was used to stain nuclei and for mounting the cells on slides. Images were acquired using a fluorescence microscope (BioZero BZ-8100; Keyence, Japan) and analysed with BZ Analyzer software (Keyence).
Cell growth analysis
HRASV12-expressing p53−/− MEFs (1 × 105) expressing each SOX2 sgRNA separately were seeded in 6-well plates. Cell numbers were determined using a Vi-CELL cell analyser (Beckman, Brea, CA, USA) on the indicated days after plating, and cell growth curves were created.
Sphere formation assay
Cells (5 × 103 or 1 × 104) were plated in 6-well, ultra-low attachment plates and grown in serum-free DMEM/F12 medium containing epidermal growth factor (20 ng/mL) and basic fibroblast growth factor (10 ng/mL) for 7 days. The numbers of spheres were counted in each treatment group. Sphere images were captured using the BZ-8100 microscope.
Colony formation assay
A layer of 1.5% (weight/volume) agarose prepared in DMEM containing 10% FBS was added to the wells of 6-well plates. Agarose (0.6%) containing 3 × 104 HRASV12-expressing p53−/− MEFs containing each SOX2 sgRNA was added to the top of the first layer. After 30 days, each well was stained with 0.005% crystal violet (Sigma-Aldrich), and the colonies were counted.
Animal experiments and cell line xenografts
The animal experiment protocol was approved by the Ethics Committee on Animal Experiments of Nippon Medical School (ethics approval number 26-020, 27-188). Animal experiments were carried out in accordance with the guidelines for Animal Experiments of Nippon Medical School and the guidelines of The Law and Notification of the Government of Japan as well as the ARRIVE guidelines. Mice were maintained at 20–24 °C in a facility with a 12 h light/ dark cycle and 40%–70% humidity. The mice were allowed free access to water and standard MF laboratory mouse chow (Oriental Yeast Co., ltd. Tokyo, Japan) and housed at a maximum number of five per cage. All mice were checked for stress each day. For the xenograft experiments, 5-week-old male BALB/cAJcl-nu/nu mice were purchased from CLEA Japan, Inc. (Tokyo, Japan) and assigned at random to the experiments. These mice were subcutaneously injected with 1 × 105 cells of p53−/− MEFs, HRASV12-expressing p53−/− MEFs or HRASV12-expressing p53−/− MEFs containing SOX2 gRNA. The number of mice used are indicated for each experiment. Tumour growth was monitored every 3 days for 3 weeks and each week thereafter by caliper measurements, and tumour size was determined using the following formula: (Length × Width2)/2. At the end of the experiments (3 or 13 weeks post-injection), mice were euthanized by cervical dislocation, then each tumour were removed and weighed, and also collected for further experiments.
All experiments were repeated at least three times independently. Data are presented as means ± standard deviation (SD). Statistical analysis was performed with the Student’s t-test using Microsoft Excel (Microsoft, DC, USA). P < 0.05 was considered statistically significant.