Spheroid growth assays as a function of glutamine, pyruvate, biotin and FBXW7.
Description
We hypothesized that 293T spheroids could model proliferation as a result of nutrient (glutamine, pyruvate, biotin) and genetic (FBXW7 KO) perturbation. Our data shows that in the absence of glutamine, pyruvate rescues proliferation only upon biotin supplementation and FBXW7 expression.
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Steps to reproduce
5,000 HEK293T cells per well were seeded on U-bottom NunclonTM SpheraTM 96-Well plates (ThermoFisher, 174925) in 200 µL of glucose-free and glutamine-free DMEM supplemented with 25 mM glucose, 10% dialyzed FBS, and 100 U/mL penicillin/streptomycin. Experimental media were supplemented with either 2 mM pyruvate or 2 mM glutamine, as indicated. The plates were then briefly centrifuged to allow cells to settle at the bottom of the well. Spheroids formed spontaneously and samples were imaged every 3-4 days with an EVOSTM FL imaging system (Life Technologies) with a 4x objective or with an Incucyte® S3 Live-Cell Analysis System (Sartorius) with a 4x objective. Spheroid diameter was quantified using Fiji (ImageJ). For biotin and pyruvate supplementation, HEK293T cells were first cultured in standard DMEM (25 mM glucose, 1 mM pyruvate, 2 mM glutamine) supplemented with 100 U/mL penicillin/streptomycin and 10% dialyzed FBS for two weeks to deprive cells of biotin. After 14 days, HEK293T cells were seeded in in glucose-free and glutamine-free DMEM containing 25 mM glucose and supplemented with 10% dialyzed FBS and 100 U/mL penicillin/streptomycin and treated as above. Test media were prepared with the addition of either 2 mM of pyruvate, 1 µM of biotin, or both, as indicated in the figure legends.