12 ham dmem f12 medium

The SH-SY5Y cell line was obtained from the American Type Culture Collection (ATCC) (Manassas, VA, USA). Cells were cultured using Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 Ham (DMEM/F12) medium (Sigma-Aldrich, Saint Louis, MO, USA), containing 10% Fetal Bovine Serum (FBS) (Sigma-Aldrich, Saint Louis, MO, USA), 1% glutamine, and 1% penicillin-streptomycin. In order to induce neuronal differentiation, SH-SY5Y cells were treated with 10 µM RA for 5 days. At the end of the 5-day-differentiation, the medium was replaced with fresh medium with or without CBN at the concentration 10 or 20 µM (dissolved in DMSO; final DMSO concentration <0.1%). A previous report showed no cytotoxicity at these concentrations [12 (link)]. After 24 h of pre-treatment, the medium was changed with fresh medium with 1 mM MPP⁺ dissolved in water for 48 h. The MPP⁺ concentration and time of exposure were chosen on the basis of a previous report [15 (link)]. Control cells were incubated with DMEM/F12 medium supplemented with 10% FBS.

Chicken Leghorn male hepatocellular (LMH) cells were maintained in Dulbecco’s modified Eagle’s medium/nutrient mixture F-12 ham (DMEM/F12) medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA) and incubated at 37 °C in 5% CO₂.
The highly pathogenic FAdV-4 strain HLJFAd15 (GenBank No. KU991797) was isolated in our previous study [19 (link)]. The artificial non-pathogenic strain rHN20 is a chimeric virus generated by replacing its hexon gene with that of ON1 (GenBank No. GU188428) using HLJFAd15 as the backbone [20 (link)]. The recombinant FAdV-4 rWT-EGFP strain expressing EGFP was derived by inserting an EGFP expression cassette at the 1966-bp site of the natural deletion in the HLJFAd15 genome. All viruses were amplified in LMH cells and stored at –80 °C. The HLJFAd15 and rHN20 viral titers were titrated with plaque-forming units (PFU) in monolayer LMH cells. The median tissue culture infective dose (TCID₅₀) was used to calculate the viral titer of rWT-EGFP according to the expression of EGFP. The titers of HLJFAd15, rHN20 and rWT-EGFP were 1.36 × 10⁷ PFU/mL, 3.14 × 10⁷ PFU/mL, and 10^7.67 TCID₅₀/mL, respectively.