The generation of an inducible single-copy C9orf72 transgene using Flp-In TRex-HeLa cells was as described previously[31 (link)]. All cell lines used in this study were maintained in DMEM (GE Healthcare HyClone, SH30243.01) with 10% FBS (GE Healthcare HyClone, SV30160.03) and 1% Pen-Strep (Thermo Fisher Scientific, 15070063) at 37°C and 5% CO2. Tetracycline (Sigma-Aldrich, T7660) was added (100 ug/ml) for 18 h to induce the expression of C9orf72 transgene. Parental HeLa cells treated with Tetracycline were used as controls in the experiments shown in Figure 6b and 7b . All plasmid transfections in cell lines were performed using Lipofectamine 2000 (Thermo Fisher Scientific, 11668019) or TurboFect (Thermo Fisher Scientific, R0531) as per the manufacturer’s instruction. For autophagy experiments, Chloroquine (CQ, Sigma-Aldrich) was dissolved in water (20 mM) and used at 40μM in the media. Autophagy induction using Earle’s Balanced Salt Solution (EBSS, Gibco) was incubated on cells following two successive PBS rinses. Incubation time for each condition is stated in the respective figure legends.
Protocol detail
Inducible C9orf72 Transgene Model in HeLa Cells
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Autophagy
Cell lines
Cells
Chloroquine
Ebss
Hela cells
Lipofectamine 2000
Parental
Plasmid
Salt
Strep
Tetracycline
Transfections
Transgene
Corresponding Organization :
Other organizations : University of Pennsylvania, National University of Singapore, Alfred Health, Newcastle University, University of California, San Francisco, Banc de Sang i Teixits, Universitat de Barcelona, Columbia University, Bank of New York Mellon, The University of Texas Southwestern Medical Center, Ludwig-Maximilians-Universität München, Johns Hopkins University, King's College London, Emory University, Indiana University – Purdue University Indianapolis, Institute for Neurodegenerative Disorders, University College London, National Hospital for Neurology and Neurosurgery, Mayo Clinic in Florida
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Variable analysis
independent variables
- Tetracycline (100 ug/ml) for 18 h to induce the expression of C9orf72 transgene
- Expression of C9orf72 transgene
- DMEM with 10% FBS and 1% Pen-Strep
- Cell lines maintained at 37°C and 5% CO2
- Positive control: Parental HeLa cells treated with tetracycline
- Negative control: Not explicitly mentioned
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