For pharmacological inhibition of aerobic glycolysis, C26 syngeneic colon cancer mice and control mice received daily intraperitoneal injections with heptelidic acid (1 mg/kg; Cayman Chemical, Ann Arbor, MI, USA), starting on day 14 post-subcutaneous injection of C26 cells.
Toxicity testing of heptelidic acid in the C26 and control mice was performed on day 4 after the start of treatment. Cardiac blood punctures were collected, allowed to clot at room temperature (15–30 min), and then the clot was removed by centrifuging at 1000–2000× g for 10 min in a refrigerated centrifuge (4 °C). The resulting supernatant sample was designated serum and was used for the toxicity tests that were performed by IDEXX Laboratories, Inc., Westbrook, ME, USA.
GAPDH activity measurements in the liver, kidney, and pancreas was performed on day 4 after the start of treatment with heptelidic acid in the C26 and control mice. Tissues were collected and kept in ice cold PBS during dissection. Lysis and homogenization of 10 mg of each fresh tissue in GAPDH Assay Buffer was performed using 5 mm Stainless Steel Beads and TissueLyser II (Qiagen, Hilden, Germany). The GAPDH activity was measured using a GAPDH Activity Assay Kit (#K680-100, BioVision, Milpitas, CA, USA).
Toxicity testing of heptelidic acid in the C26 and control mice was performed on day 4 after the start of treatment. Cardiac blood punctures were collected, allowed to clot at room temperature (15–30 min), and then the clot was removed by centrifuging at 1000–2000× g for 10 min in a refrigerated centrifuge (4 °C). The resulting supernatant sample was designated serum and was used for the toxicity tests that were performed by IDEXX Laboratories, Inc., Westbrook, ME, USA.
GAPDH activity measurements in the liver, kidney, and pancreas was performed on day 4 after the start of treatment with heptelidic acid in the C26 and control mice. Tissues were collected and kept in ice cold PBS during dissection. Lysis and homogenization of 10 mg of each fresh tissue in GAPDH Assay Buffer was performed using 5 mm Stainless Steel Beads and TissueLyser II (Qiagen, Hilden, Germany). The GAPDH activity was measured using a GAPDH Activity Assay Kit (#K680-100, BioVision, Milpitas, CA, USA).