Glucose glotm assay

The concentration of glucose in medium was assayed using a Glucose-Glo^TM assay (Promega, Madison, WI, United States) according to the manufacturer’s instructions.

Intracellular metabolites were measured using Glucose-Glo^TM Assay, Lactate-Glo^TM Assay, and Glutamate-Glo^TM Assay (Promega) according to the manufacturer’s protocol.

To study glucose production through gluconeogenesis, McA-RH7777 cells were cultivated under appropriate conditions. Cells were treated with CK2 inhibitor or transfected with siRNA as described above. The night before the assay, DMEM high-glucose medium (4.5 g glucose/L) was replaced by low-glucose medium (1 g glucose/L). The next day, the medium was withdrawn and cells were washed twice with Krebs-Ringer buffer KRB (120 mM NaCl, 5 mM KCl, 2 mM CaCl₂, and 1 mM MgSO₄) to remove any residual glucose. Cells were incubated for another 2 h with KRB. Gluconeogenesis was induced by the addition of 10 mM lactate and 10 µM forskolin in KRB. After one hour incubation at 37 °C, supernatants were collected and secreted glucose was determined with the bioluminescent Glucose-Glo^TM Assay from Promega (Mannheim, Germany) according to the recommendations of the manufacturer. Luminescence was recorded in an Infinite M200 Pro TECAN Reader (Sigma-Aldrich, München, Germany) using the luminescence mode.