D α tocopherol polyethylene 1000 succinate

Athymic nude mice (Crl; Nu-Foxn1^Nu/Nu) were implanted with 2 million TC32 cells in the left gastrocnemius muscle in a volume of 100 μL Hank’s Balanced Salt Solution (Thermo-Fischer Scientific). Once tumor diameter reached 0.5 cm, mice were randomly assigned to either the control, mithramycin, PHA-767491, or combination groups. Mithramycin was administered via an Alzet 1003D osmotic pump (Durect) placed within the peritoneum which delivered a dose continuous dose in a vehicle of DPBS with MgCl₂ and CaCl₂ (MilliporeSigma) and was removed after treatment. PHA-767491 was administered via oral gavage in 200 μL of its vehicle, 1 mg/10 mL D-α-Tocopherol polyethylene 1000 succinate (MilliporeSigma). Mice were euthanized by CO₂ asphyxiation when tumor diameter reached greater than 2 cm³. Tumor size was determined by the equation( $\frac{D{d}^2}{6}*3.12$ ), where D is the maximum diameter and d is the minimum diameter. All animal work was approved by the Van Andel Institute Animal Care and Use Committee.

Mithramycin (Tocris Bioscience) was dissolved as a 1 mg/mL stock solution in PBS, and frozen in aliquots. PHA-767491 and other small molecule inhibitors (Selleck Chemicals) were dissolved in DMSO as stock solutions. Small molecule stocks were stored at −80 °C for storage or −20 °C for experimental use, minimizing freeze-thaw cycles. For in vivo experiments, a 30 mg/mL stock solution of PHA-767491 (Adooq Bioscience) was made by dissolving the powder in a solution of 1 mg/10mL D-α-Tocopherol polyethylene 1000 succinate (MilliporeSigma) for oral gavage.