Hela s3

Hela S3 and VERO E6 cell lines were purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, China) and cultured in DMEM medium (Gibco, Grand Island, NY, USA, 11965118) supplemented with 10% FBS (Gibco, 10091148) and 1% penicillin and streptomycin (Gibco, 15070-063), respectively. Vaccinia virus (ATCC-VR-1354) was purchased from ATCC (Manassas, VA, USA) and grown in Hela S3 and VERO E6. The virus titer was determined by VERO E6-based plaque assay. Monkeypox virus (IVCAS 6.9141, Clade IIb) was isolated from a patient in Wuhan (China) and propagated in VERO E6 cells prior to MPXV neutralizing assay.

HeLa, HeLa S3 and 293T were purchased from the ATCC. Mfn1^−/− and WT MEFs have been previously described.^{4 (link)} MARCH5-knockout HeLa and 293 cells were generated by using transcription activator-like effector nucleases technology. The MARCH5-specific transcription activator-like effector nuclease plasmids were obtained from ToolGen Inc. (Seoul, Korea).^{40 (link)} MARCH5^−/− MEFs were generated from C57BL/6 MARCH5^flox/flox embryo. Cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% heat-inactivated fetal bovine serum, 1% penicillin and streptomycin (Gibco, BRL, Grand Island, NY, USA) in a 5% CO₂ incubator at 37 °C. Plasmid DNA constructs were transfected by using polyethylenimine (Polysciences, Inc., Warrington, PA, USA) as previously described.^{19 (link)} For shRNA, 1 day after transfection, cells were grown in the media containing 200 μg/ml hygromycin B (Roche Diagnostic, Indianapolis, IN, USA) for 36 h to select transfected cells. Dying cells were removed by brief centrifugation and the survived cells were re-seeded on culture plates, which is designated as day 0 and further grown in 30 μg/ml hygromycin B containing Dulbecco's modified Eagle's medium.^{19 (link)}

The adherent human adenocarcinoma cell line A549 (ATCC CCL-185) and human cervix adenocarcinoma cell line HeLa S3 (ATCC CCL-2.2) were obtained from ATCC and cultured as monolayer in DMEM containing 10% FCS, 100 U/mL penicillin and 100 mg/mL streptomycin. Human lung bronchial epithelial BEAS-2B cells (ATCC CRL-9609), immortalized with SV40 large T-antigen, were kindly provided by Dr. Carsten Weiss (Karlsruhe Institute of Technology, Karlsruhe, Germany). They were grown as monolayers in coated cell culture dishes (10 μg/mL human fibronectin, 30 μg/mL collagen and 10 μg/mL bovine serum albumin in PBS) in LHC-9 medium containing 2.5 μg/mL amphotericin B. Cells were incubated at 37 °C in a humidified atmosphere of 5% CO₂ in air. For all experiments cells were seeded at a density of 16.600 cells/cm². After 24 h of cultivation in case of A549 and HeLa S3 cells and 48 h in case of BEAS-2B cells the supernatant from the logarithmically growing cells was removed and replaced by the particle and CuCl₂ incubation suspensions or dilutions (0.2 mL/cm²) in DMEM containing 10% FCS independent from the respective cell culture medium to ensure comparable incubation conditions for all applied cell lines.

All cell culture media and supplements were purchased from Life Technologies (Eugene, OR). The human cell line Hela S3 was purchased from ATCC (Manassas, VA) and maintained in Dulbecco’s Modified Eagle Medium (DMEM), high glucose plus GlutaMAX, containing 10% FCS (Atlas Biologicals, Fort Collins, CO). NFAT-RE-luc2 Jurkat cells (Promega, Madison, WI) were cultured in RPMI-1640 containing 10% FCS, MEM non-essential amino acids (1x), sodium pyruvate (1 mM) and hygromycin B (200 μg ml⁻¹). All cell lines were maintained at 37 °C under 5% CO₂.

HeLa S3, HT1080, U-2OS, BJ, 293T and Phoenix AMPHO (ATCC, Rockville, MD) were cultured in Dulbecco’s modified Eagle medium (DMEM) (Corning) supplemented with 10% fetal bovine serum (FBS) (Gibco), l-glutamine (Gibco), non-essential amino acids (Gibco) and penicillin–streptomycin (Gibco) at 37°C and 5% CO₂. Bortezomib (PS-341; Selleckchem) was dissolved in DMSO (Sigma) and added to the cells (final concentration of 100 nm) for 6 h before harvest.