Lactate assay kit

Antibodies against GST (1:1000; Z-5, B-14, Santa Cruz), FLAG (1:1000; M2, Sigma), V5 (1:1000; D3H8Q, Cell Signaling), CAD (1:1000; A301-374A, Bethyl Laboratories), RelA (1:1000; 51-0500, Thermofisher), Pyruvate Carboxylase (1:1000; #66470, Cell Signaling, #49381, Cell Signaling), PDK3 (1:1000; AP7040a, Abcepta), Hexokinase 4 (1:1000; AP7901c, Abcepta), β-Actin (1:2000; 8H10D10, Cell Signaling), Phospho-CAD (1:1000; Ser1859, Cell Signaling), LANA (1:500; LN53, Abcam), vCyclin (1:500; 94B, Abcam), MYC(1:1000; 71D10, 9B11, Cell Signaling), CDK6 (1:1000; D4S8S, Cell Signaling) were purchased from the indicated suppliers. Polyclonal antibodies against KSHV vCyclin (1:500) and Phospho-CAD (Ser1900, 1:500) were generated by Genscript.
The glutamine analog 6-Diazo-5-oxo-L-norleucine (DON) was purchased from Sigma. JHU-083 (Ethyl 2-(2-Amino-4-methylpentanamido)-DON), Palbociclib, and N-(phosphonacetyl)-L-aspartic acid (PALA) were purchased from MedChemExpress. [U¹³C] Glucose and [Amide-¹⁵N] L-Glutamine were purchased from Cambridge Isotope Lab. Luciferin was purchased from Goldbio.
L-Lactate Assay Kit was purchased from Cayman Chemical. Cell proliferation Kit II, and Glutamate Assay Kit were purchased from Sigma. SYBR Green Master Mix was purchased from Bio-Rad and Genesee. Dual-Luciferase Reporter Assay System was purchased from Promega.

Lactate and glucose assays were performed by utilizing commercially available kits from Cayman Chemicals (lactate assay kit: catalogue No. 600450 and glucose assay kit: catalogue No. 10009582). Cells were seeded as per the manufacturer’s instructions in the kit, and media of cells was used to determine the amount of lactate production and glucose consumption upon termination of the experiment at 24 and 48 h. Further, the absorbance was recorded at the indicated wavelength, and readings were analysed/calculated.

Lactate and glucose assays were performed using kits from Cayman Chemicals (Lactate assay kit catalog number: 600450, Glucose assay kit catalog number: 10009582). MUC13-expressing and MUC13-null cells were seeded (10⁴ cells/well in 96-well plate) and media collected to measure the amount of lactate after 24 h, and unused glucose levels after 48 h. The samples were analyzed according to the instructions provided in the kit, the readings were recorded and calculations were done.

The assays were carried out according to the protocol of glucose uptake assay kit (colorimetric, Abcam) and lactate Assay Kit (Cayman Chemical).

RCC cells were seeded in a 6-well plate (1 × 10⁶/well) and incubated at 37°C for 24 hours under indicated treatment. The culture medium was collected for following testing. Glucose and lactate levels were determined using the Glucose (GO) Assay Kit (Sigma) and the Lactate Assay Kit (Cayman Chemical), respectively. Intracellular lactate level was determined by LC–MS system (Thermo Fisher TSQ). For testing the excised tumor, the tumor tissue from each animal were homogenized and suspended (50 mg/ml) in ice-cold buffer containing 10 mM Tris HCl (pH 7.5), 1 mM EDTA, 10 μg/ml leupeptin, 11.5% sucrose, 10 μg/ml pepstatin A, 10 μg/ml aprotinin. The total protein concentration was measured by NanoDrop 2000 (Thermo Scientific). The final glucose and lactate values were normalized to the total protein concentration.