Cell culture and drug treatment protocol

Cells were grown in DMEM (high glucose) supplemented with penicillin and streptomycin, 10% FCS and were cultured at 37 °C and 5% CO₂. U2OS cells were purchased from ATCC and HeLa cells were kindly provided by M. Kühl and were purchased from Sigma (Steinheim, Germany). HEK293A GFP-LC3 cells (ECACC 14050801) were purchased from Sigma (Steinheim, Germany) and were cultured in medium supplemented with 0.4 mg/mL G418 (Sigma, Steinheim, Germany) [34 (link),40 (link)]. Cell lines were regularly tested for absence of mycoplasma and were used in low passage numbers.
HeLa and U2OS cells were seeded in 6-wells and were transfected with 45 nM si RNAs for 48 h using Oligofectamine according to the manufacturer´s instructions. For drug treatment, cells were seeded in 6-wells and grown over night prior administration of DMSO (Sigma) or 1 µM CX-5461 (Caymen, Ann Arbor, MI, USA) for 24 h as indicated. For flux assays in HEK293A GFP-LC3, cells were incubated with DMSO or 1 µM CX-5461 for 20.5 h followed by 50 µM chloroquine (CQ; Sigma, Steinheim, Germany) for 3.5 h.