Dmem high glucose medium

The human Jurkat T cell (subclone E6-1) and its Lck kinase deficient derivative, J.Cam1, were obtained from American Tissue Type Collection. Three HCV C protein-expressing Jurkat T cell lines (JHC.d, JHC.g and JHC.h) contain DNA sequence from the infectious HCV H77 strain encoding the first 194 amino acid of the HCV polyprotein as described previously [17 (link),18 (link)]. Single cell clones were selected on puromycin (0.2 µg/mL final concentration) and tested for the C protein expression by western blotting. Human monocytic THP-1 cell line was kindly provided by Dr. Göran Akusjärvi, Uppsala University, Uppsala, Sweden. The human embryonic kidney cell line HEK293TT expressing high levels of SV40 large T-antigen was kindly provided by Dr. Helena Faust [44 (link)]. The Jurkat, J.Cam1, JHC.d, JHC.g, JHC.h (collectively as JHC) and THP-1 cell lines were cultured in RPMI-1640 media (Sigma-Aldrich) supplemented with 10% fetal calf serum (FCS) and 1% penicillin-streptomycin mixture (Sigma-Aldrich) whereas 293TT cells were cultured in DMEM (high glucose) medium (Sigma-Aldrich) with 10% FCS and 1% penicillin-streptomycin mixture at 37 °C in a 5% CO₂ humidified atmosphere.

Mouse embryonic stem cell line (kind gift from Ivan Bedzhov, MPI Münster) were maintained under 5% CO₂ and 37 °C conditions on 10% gelatine-coated (Sigma, G1393) T25 flasks in DMEM high glucose medium (Sigma D5671) containing 15% FBS (Sigma S0615), 100 U/ml Penicillin-Streptomycin, 2 mM L-Glutamine, 1 mM sodium pyruvate (Sigma, S8636), 0.1 mM NEAA (Sigma, M7145), 0.1 mM 2-mercaptoethanol (Sigma, M3148). Maintenance medium was freshly supplemented with 0.44 nM mLif (Amsbio, AMS-263), 0.4  $\mu$ M PD 0325901 and 3  $\mu$ M CHIR 99021 inhibitors (Cayman Chemicals). Seeding cell number was set to 4.2 × 10⁵ cells (for 2-day interval passaging) or 1.5 × 10⁵ (for 3 day interval passaging). The cells carry a fluorescent marker in the nucleus (H2B–GFP⁺) and express a red Ca²⁺ sensor (R-GECO 1.0). The cell line was detected positive for myoplasm after the experiments were performed.

The human bronchial epithelial BEAS-2B cells were purchased from America Type Culture Collection (ATCC). The arsenic-transformed cells were described previously 30 (link). The rat inslinoma cell line INS1 was a gift from Dr. Anjaneyulu Kowluru, Professor of Pharmaceutical Sciences, Wayne State University. All cells were cultured in DMEM-high glucose medium (Sigma, cat.no. D5796) supplemented with 5% FBS, 1% penicillin-streptomycin (Gibco, cat.no. 15140122), and 1% L-Glutamine (Gibco, cat.no. 25030164). Cells were maintained in a humidified incubator at 37 °C with 5% CO₂. In some experiments, the cells were treated with 0.25 to 2 μM arsenic as reported previously 30 (link).

VeroE6/TMPRSS2 cells (1.8 × 10⁵) were seeded in a 24-well plate with culture medium (DMEM high glucose medium supplemented with 10% FCS, 1% L-Glutamine, 1% Penicillin/Streptomycin; all reagents were obtained from Sigma Aldrich, Missouri, USA) and incubated overnight at 37°C and 5% CO₂. On the following day, whole serum or plasma samples were serial-diluted from 1:1 to 1:6,250 and incubated with SARS-CoV-2 strains (1.5 × 10⁴ PFU/ml) for 1 h at 37°C. After incubation serum/plasma-virus mix was ultracentrifuged and resuspended in DMEM high glucose medium supplemented with 5% FCS, 1% L-Glutamine, 1% Penicillin/Streptomycin. VeroE6/TMPRSS2 cells were then inoculated with antibody-opsonized SARS-CoV-2 for 30 min on a shaker at RT and 30 min in an incubator at 37°C and 5% CO₂. After incubation, inoculation medium was replaced with culture medium containing 1.5% Low-melt Agarose (Biozym, Oldendorf, Germany). Cells were incubated for 3 days at 37°C and 5% CO₂ before plaque visualization and counting using 0.1% Neutral Red solution for 3 h (Sigma Aldrich, Missouri, USA).

Zn(NO₃)₂, C₄H₆N₂, Folic acid, Mueller Hinton agar, Nutrient agar, penicillin, streptomycin, Fetal bovine serum, DMEM high glucose medium, Crystal violet, Artemia cysts and Methyl gallate were purchased from Sigma Aldrich, Darmstadt, Germany. All the chemicals and reagents were used without purification.

MCF7, T47D, SKBR3 and HeLa cell lines were obtained from the American Type Culture Collection (Rockville). A4-HeLa was kindly provided by Dr. Tom Jovin and Dr. Donna Arndt-Jovin (Max Planck Institute for Biophysical Chemistry, Göttingen, Germany). MCF7 and T47D cell lines were routinely maintained in DMEM/F12 cell culture medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS, Internegocios, Córdoba, Argentina) in a humidified 5% CO2/air atmosphere. SKBR3 cell line was cultured in RPMI medium (Sigma-Aldrich) supplemented with 10% FBS. HeLa and A4-HeLa cell lines were maintained in DMEM high glucose medium (Sigma-Aldrich) supplemented with 10% FBS. Serial passages were carried out by treatment of 80% confluent monolayers with 0.25% trypsin (Invitrogen) and 0.02% EDTA in Ca2+-free and Mg2+-free phosphate-buffered saline (PBS).