On day 0, 100,000 NT or GRAMD1B shRNA SKMEL2 cells were pretreated in 4 μg/mL dox-containing media in 6-well dishes to induce GRAMD1B knockdown for 48 hours. On day 2, cells were switched to either dox-containing control media (DMEM, 10% FBS, 1% antibiotics, 4 μg/mL dox) or cholesterol depletion media (DMEM, 10% lipoprotein-free FBS, 1% antibiotics, 10 μmol/L simvastatin, and 200 μmol/L mevalonic acid, 4 μg/mL dox) for another 48 hours. Because the mevalonate pathway is critical for cell viability, mevalonate supplementation was included in the cholesterol depletion mixture as done in Trinh and colleagues (121 (link)). On day 4, cholesterol depletion was confirmed through Promega's cholesterol Glo assay, and cells were seeded for invasion assays.
Protocol detail
GRAMD1B Knockdown and Cholesterol Depletion
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Antibiotics
Assays
Cell viability
Cells
Cholesterol
Dishes
Lipoprotein
Mevalonate
Mevalonic acid
Shrna
Simvastatin
Corresponding Organization : Memorial Sloan Kettering Cancer Center
Other organizations : Wellcome Sanger Institute, European Bioinformatics Institute, Tri-Institutional PhD Program in Chemical Biology, Cornell University, Newcastle University, University of Leeds
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Variable analysis
independent variables
- Cholesterol depletion media (DMEM, 10% lipoprotein-free FBS, 1% antibiotics, 10 μmol/L simvastatin, and 200 μmol/L mevalonic acid, 4 μg/mL dox)
- Dox-containing control media (DMEM, 10% FBS, 1% antibiotics, 4 μg/mL dox)
- Cholesterol levels (confirmed through Promega's cholesterol Glo assay)
- Invasion assays
- Cell line: SKMEL2 cells
- Initial cell seeding: 100,000 NT or GRAMD1B shRNA SKMEL2 cells
- Pretreatment: 48 hours in 4 μg/mL dox-containing media to induce GRAMD1B knockdown
- Dox concentration: 4 μg/mL in both control and cholesterol depletion media
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