We used human HeLa cells (ATCC CCL-2), MEFs, Atg5 KO MEFs (Kuma et al., 2004 (link)), and CRISPR-Cas9–generated HeLa ATG7 KO cells (Mejlvang et al., 2018 (link)). All cells were cultured in DMEM (D6046; Sigma-Aldrich) supplemented with 10% FBS (S0615; Biochrom) and 1% streptomycin-penicillin (P4333; Sigma-Aldrich) and kept in a humidified incubator at 37°C and 5% CO2. Starvation experiments were conducted by incubating cells in HBSS (H9269; Sigma-Aldrich). Cells were treated with 1 µg/ml tetracycline (Sigma-Aldrich), 200 ng/ml BafA1 (sc-201550; Santa Cruz Biotechnology), and 25 µM MG132 for the indicated time periods. DNA transfection was done with Metafectene Pro (T040; Biontex) according to the manufacturer’s protocol. siRNA transfections were done with RNAiMAX according to the manufacturer’s protocol.
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Autophagy Regulation in Cell Lines
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Cells
Crispr
Hbss
Hela cells
Human
Metafectene
Mg132
Penicillin
Sirna
Streptomycin
Tetracycline
Transfections
Corresponding Organization : UiT The Arctic University of Norway
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Variable analysis
independent variables
- Tetracycline treatment (1 µg/ml)
- BafA1 treatment (200 ng/ml)
- MG132 treatment (25 µM)
- Starvation by incubating cells in HBSS
- Cellular response to the treatments and starvation
- Cell culture media (DMEM with 10% FBS and 1% streptomycin-penicillin)
- Cell types (human HeLa cells, MEFs, Atg5 KO MEFs, and CRISPR-Cas9–generated HeLa ATG7 KO cells)
- Incubation conditions (37°C, 5% CO2, humidified)
- DNA transfection method (Metafectene Pro)
- SiRNA transfection method (RNAiMAX)
- Not explicitly mentioned
- Not explicitly mentioned
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