Athymic nude mice

Pig ESCs at 70%–80% confluence were used for transplantation. Approximately 5–10 × 10⁶ ESCs were resuspended in 200 μL of ESC culture medium containing 50% (v/v) BD Matrigel Matrix (BD Biosciences, Franklin Lakes, NJ, USA) and 10 μM Y-27632. Next, the resuspended pig ESCs were injected subcutaneously into 5-week-old athymic nude mice (OrientBio). At 2–3 months after transplantation, 1- to 2-cm teratomas were collected, fixed in 4% (w/v) paraformaldehyde, embedded in paraffin, and stained with H&E for light microscopic examination.
For genotyping of teratomas, genomic DNA was extracted from teratomas and the peritoneum of athymic nude mice using the G-spin Total DNA Extraction Kit (iNtRON Biotechnology, Korea). Genomic DNA samples were amplified using 10 pmol of species-specific primers (Table S4) and 2× PCR master mix solution (iNtRON Biotechnology). PCR reactions were performed in a thermocycler under the following conditions: 95°C for 5 min; followed by 25 cycles of denaturation at 95°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s; with a final extension at 72°C for 7 min. Amplified PCR products were electrophoresed in 1% (w/v) agarose gels and stained with RedSafe Nucleic Acid Staining Solution (an alternative to ethidium bromide; iNtRON Biotechnology).

Four-week-old female athymic nude mice were purchased from Orient Bio Inc. (Seongnam, Korea). Capan-1 cells were resuspended at 3×10⁷ cells in 100 μL of phosphate-buffered saline and injected subcutaneously. The tumor volume was calculated using the formula: volume=[(width)²×height]/2. When the tumor volume reached 200 mm³, the mice were randomly assigned to four groups of five mice to receive (1) vehicle (2-hydroxypropyl-β-cyclodextrin solution), (2) AZD1775 once daily at 30 mg/kg for 4 weeks (5 days on/2 days off), (3) AZD0156, as described for (2), or (4) AZD1775 plus AZD0156, as descri-bed for (2). All treatments were administered by oral gavage. Body weights and tumor sizes were measured every other day.