Glucose glo assay

From isolated CD4⁺ T cells and monocytes, 30 000 cells were used for metabolite detection in duplicates for each sample. Lactate-Glo assay (Promega, #J5022; Promega, Madison, Wisconsin, USA), Glutamate-Glo assay (Promega, #J7022), and Glucose-Glo assay (Promega, #J6022) were used to measure intracellular metabolites according to the manufacturer's instructions. Luminescence was measured using Varioskan microplate reader (ThermoFisher Scientific, Waltham, Massachusetts, USA).

Alteration in cellular metabolism regarding glycolysis was measured using the Glucose-Glo ™ Assay and Lactate-Glo ™ Assay (Promega, Madison, WI, USA). The cells at densities of 1 × 10⁴/well (A2780^cisR cells) and 5 × 10³/well (SNU-8^cisR cells) were seeded in 96-well plates and incubated overnight. The cells were treated with isoalantolactone (10 μM), followed by cisplatin (2 μg/mL for A2780^cisR cells and 5 μg/mL for SNU-8^cisR cells) for 24 h. The culture and control media were collected and incubated with glucose detection reagent and lactate detection reagent for 1 h, respectively. The luminescence intensity was measured using a microplate luminometer (Molecular Devices). Glucose consumption and lactate production were calculated by subtracting from the concentrations of glucose and lactate in the control medium.

The analysis of the glucose concentration in the culture medium was performed using the Glucose-Glo™ Assay (Promega, Madison, WI, USA). Briefly, the cells were seeded in 96-well plates (10⁴ cells per well) and left for 24-h preincubation. Then, the appropriate growth medium was replaced with fresh medium containing IC25 concentrations of the glycolysis inhibitors or their combinations with Wnt signaling inhibitors, and the cells were incubated for an additional 48 h. Cells incubated with solvent served as a negative control. Afterwards, 2 µL of medium from each well was diluted in 1998 µL of PBS buffer. Luminometric analysis of the glucose concentration was performed on a GloMax^® Discover microplate reader (Promega, Madison, WI, USA) after 60 min incubation of diluted culture medium (50 µL per well) with a glucose detection reagent (50 µL per well). The glucose concentration in the media was determined based on a linear standard curve, which was prepared in parallel (Supplementary Figure S1). Glucose consumption was calculated as the difference between glucose concentration in cell-free media and media samples obtained after 48 h of cell incubation in the presence of the compounds. Data were normalized based on the viability assay results. The assay was performed in triplicate, each time with three replicates per assay.

Analysis of intracellular glucose and lactate quantities were performed by using Glucose-Glo assay and Lactate-Glo Assay, respectively (Promega, Madison, WI, USA). Briefly H460 and MCF7 cells were seeded in 6-well plates and then transfected with the two siRNAs specific for ALDOC and ENOs. After transfection, both cell lines were cultured in 96-well ultra-low-attachment plates. Analysis was performed through GloMax Explorer Luminometer (Promega). Data were normalized to cell number. Analyses were performed in triplicate and results are reported as mean ± SD.

Primary MSC (0.25 × 10⁶) were treated with melanoma conditioned media or control media for 48 h. Primary MSC (0.25 × 10⁶) were treated PGC1a-KD virus or control-KD virus for 72 h. Promega Glucose Uptake Assay was performed on treated cells and Promega Glucose-Glo Assay was performed on media from treated cells according to manufacturer’s instructions.

Glucose contents in cell lysates were quantified using the Glucose-Glo Assay (Promega, Madison, WI), according to the manufacturer’s instructions. This assay combines glucose oxidation and NADH generation to produce a luminescence signal proportional to the glucose concentration. Briefly, the cell lysates were incubated with Glucose Detection Reagent for 1 hr at room temperature, then the luminescence was measured by SpectraMax M5 plate reader (Molecular Devices, San Jose, CA).