S1820

Chinese hamster ovary cells (CHO) were cultured in F12 medium (GIBCO 11765) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Biowest S1820), 100 U/ml penicillin, and 100 μg/ml streptomycin (Sigma-Aldrich P4333) at 37 °C in a humidified atmosphere of 5% CO₂. Cells were cultured to approximately 80% confluence in 6-well culture plates. The pcDNA3.4-TOPO^Gc1F-His expression plasmid (2.5 μg) and Lipofectamine 2000 (15 μl) (ThermoFisher Scientific Co.) were added in 2 ml medium per well. The plates were incubated for 3 days, then the culture media and cells were harvested. The expression level and sugar modification of Gc1F-His were evaluated.

The human colorectal cancer cell line DLD-1 was cultured in Roswell Park Memorial Institute-1640 medium (R8758; Sigma-Aldrich Corp., St. Louis, MO, USA) with 10% fetal bovine serum (FBS; S1820; Biowest SAS, Nuaillé, France) and 1% penicillin/streptomycin (26253-84; Nacalai Tesque, Inc., Kyoto, Japan) at 37 °C. HEK293T cells and Hela cells were cultured in Dulbecco’s Modified Eagle’s Medium (high glucose) (D5796; Sigma-Aldrich Corp., St. Louis, MO, USA) with 10% FBS and 1% penicillin/streptomycin at 37 °C. Cells were transiently transfected with siRNAs using Lipofectamine^® RNAiMAX™ (13778150; Invitrogen, Carlsbad, CA, USA), as indicated by the manufacturer. DLD-1 cells were transiently transfected with plasmid vectors using Lipofectamine^® LTX & Plus™ (15338-100; Invitrogen) or Lipofectamine^® 3000 (L3000015; Invitrogen). DLD-1 cells were co-transfected with an siRNA and a plasmid vector for the cell-cycle assay using Lipofectamine^® 3000 (L3000075; Invitrogen), as indicated by the manufacturer. DLD-1 cells were treated with CHX (06741-91; Nacalai Tesque, Inc.).

A TM4 (#88111401, ECACC, Salisbury, Wilts, UK) SC line was cultured in a Dulbecco’s Modified Eagle’s medium (DMEM)/F-12 nutrient mixture (D8437, Sigma-Aldrich, St. Louis, MO, USA) containing 5% horse serum (MIC2921149, MP Bio, Tokyo, Japan) and 2.5% fetal bovine serum (FBS) (S1820, Bio West, Nuaille, France) at 37 °C in 5% CO₂. The cells were treated with ethanol (50 or 100 mmol/L,) with and without autophagy inhibitor 3-MA (sc-205596, Santa-Cruz Biotechnology, TX, USA) as reported by others [41 (link),44 (link)].

Porcine aortic tissues were obtained from a local slaughterhouse. VSMCs were isolated from porcine aortic tissues using an explant method described in detail by Nagayama et al.^{38 (link)}. The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% foetal bovine serum (S1820, Biowest) and 1% penicillin-streptomycin at 37°C in 5% carbon dioxide and 95% air.
Retardations of passage 2 and 12 cells were compared because lower passage VSMCs have a contractile phenotype, while higher passage VSMCs dedifferentiate towards the synthetic phenotype^{39 (link)}.