HEK293T cells were cultured in high-glucose (4.5g/l) DMEM medium supplemented with 10% fetal bovine serum (FBS) and 1% sodium pyruvate. Stress experiments were conducted in 96 well plates for cell viability assay by MTT or in 6 well plates or 10cm dishes when samples were collected for western blotting or sequencing. Cells were seeded 24 hours before the stress experiment and cultured either in regular media, or in serum free media with or without 1μM Queuine supplementation (Toronto research, Cat# Q525000) Cells were stressed by Arsenite (sodium metaArsenite), Rotenone (Respiratory complex I inhibitor), Thenoyltrifluoroacetone (TTFA; Respiratory complex II inhibitor), Antimycin A (Respiratory complex III inhibitor), potassium cyanide (KCN; Respiratory complex IV inhibitor), or Oligomycin (Respiratory complex V inhibitor) for the indicated doses and duration. Cell proliferation was conducted by passaging the cells at low concentrations into 96 wells or 6 wells plates (10,000 cells per well) and analyzing the cells over time using MTT or by cell counting by Trypan blue respectively.
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Metabolic Stress Response in HEK293T Cells
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Corresponding Organization : Tohoku University
Other organizations : Massachusetts Institute of Technology, Teikyo University, Singapore-MIT Alliance for Research and Technology, Tohoku University Hospital, University at Albany, State University of New York
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Variable analysis
independent variables
- Stress treatments: Arsenite, Rotenone, Thenoyltrifluoroacetone (TTFA), Antimycin A, potassium cyanide (KCN), Oligomycin
- Culture media: Regular media, serum free media, serum free media with 1μM Queuine supplementation
- Cell viability (measured by MTT assay)
- Protein expression (measured by western blotting)
- Gene expression (measured by sequencing)
- Cell line: HEK293T
- Cell culture conditions: High-glucose (4.5g/l) DMEM medium supplemented with 10% fetal bovine serum (FBS) and 1% sodium pyruvate
- Cell seeding density: 10,000 cells per well in 96-well or 6-well plates
- Positive control: Not explicitly mentioned
- Negative control: Regular media (no stress treatment or Queuine supplementation)
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