M7145 100ml

Wildtype HeLa cells were seeded onto 12 mm glass coverslips in a density of 100.000 cells/24 well. The cells were kept in HeLa cell medium consisting of 10% FBS (S 0615; Biochrom), 1% Hepes (15630106; Thermo Fisher); 1% Non-Essential Amino Acids (M7145-100ML; Sigma Aldrich), 1% L-Alanyl-Glutamine (03-022-1B; Neo-Lab) and 1% Penicillin/Streptomycin (P4333-100ML; Sigma Aldrich) in DMEM. The next day, the cells were transfected with HCN4-pEGFP and with either CVB3-2C/pIRES-dsRed-Express2 or CVB3-3A/pIRES-dsRed-Express2 under the use of Lipofectamine3000 Transfection reagent (L3000001, Thermo Fisher). Pharmacological treatment was started directly after transfection. The HeLa cells were either treated with 0.1% DMSO; 2.5 μM N6-Benzyladenosine (4294-16-0; Santa Cruz); 10 μM GW5074 (1381, Tocris) or 1 μM CID 1067700 (ML282; Axonmedchem) for 8 h, 12 h or 24 h. The cells were immunostained against HCN4, Rab7 and LC3, as described before, and imaged. Colocalization analysis was performed with the JACoP plugin for ImageJ. Obtained Pearson´s coefficients (r), which describe the grade of localization, were multiplied with themselves to generate (r²) which was then used to give a percentual colocalization statement [26 (link), 29 (link)].

Mixed glia culture were prepared from the published protocol by Bronstein et al.⁷⁸ (link) Cortices from 3-5 murine pups aged P0-P2 of mixed sex were dissected under a sterile ventilation hood in ice-cold Hank’s buffered saline (ThermoFisher, #14025-050) and digested in 0.05% (v/v) Trypsin + 1X EDTA (ThermoFisher, #25300-054) for 15 minutes at 37°C. The trypsinization was stopped with the addition of serum-containing medium (DMEM, 10% (v/v) heat-inactivated FBS, 1% (v/v) Penicillin/Streptomycin, 1% (v/v) Non-essential amino acids (Sigma-Aldrich, M7145-100ML)). Cells were pelleted at 500xg for 5 min, washed one time with serum-containing medium, then passed through a 40μm cell strainer (Szabo-Scandic, #352340). For Western blotting, the resulting cell suspension was plated onto T75 cell culture flasks (TPP, #Z707503) and maintained at 37°C and 5% CO₂ in a humidified incubator. The culture medium was replaced at day 3 and day 10.