Dulbecco modified eagle medium (dmem)

HeLa cells (American Type Culture Collection; ATCC) were cultured in DMEM (Biowest, Riverside, MO, USA) supplemented with 10% FBS (Welgene, South Korea) and 1% antibiotic antimycotic solution, 100× (Corning, Manassas, VA, USA). Transfection was executed using Lipofectamine 2000 reagent (Invitrogen, Grand Island, NY, USA) according to the manufacturer’s instructions. The transfected cells were cultured for 24–48 h, washed with DPBS, and harvested with lysis buffer (Invitrogen, Grand Island, NY, USA). C2C12 cells were a gift from Yong-Gyu Ko at Korea University in South Korea. C2C12 cells were cultured in DMEM supplemented with 10% FBS and 1% antibiotic antimycotic solution, 100× (Biowest, Riverside, MO, USA). For induction of differentiation, fully confluent C2C12 cells were cultured in DMEM supplemented with 2% horse serum (Sigma-Aldrich, MO, USA) for 3 days.

To demonstrate the labeling efficiency of SPIONs-C595 nanoparticle, MCF-7 cells were cultured at 1×10⁵ cells/ml on each well of a 12-well plate in 1 ml serum-free DMEM (Biowest, France), 100% humid atmosphere with 5% CO₂ at 37ºC for 24 h. After the cells were 80% confluent, the 25, 50, 100, and 200 μg Fe/ml doses of SPIONs-C595 were prepared in 1 ml DMEM (Biowest, France). Subsequently, different concentrations of nanoprobe were added to the cells and the incubation was continued for 6 h. Duplicate samples were prepared for each concentration. The control groups, the MCF-7 cells, were not treated by nanoprobe. The supernatant was removed after 6-h incubation, and MCF-7 cells were washed two times with 200 μl PBS in order to remove nanoparticles that were free in solution or loosely adhered to cell surface. The cells were then trypsinized with 200 μl Trypsin. Subsequently, 1 ml 50% nitric acid was added to the cells, and the cell suspension was left in the incubator (Binder, Singapore) to further digest for 24 h. The samples were diluted up to 10 ml with deionized water for measuring the iron content of the treated and untreated cells with anatomic absorption spectrophotometer (AAnalyst 400, USA).

All experiments were conducted on the MDA-MB-468 breast cancer cell line (estrogen receptor (ER)-negative, progesterone receptor (PR)-negative, and HER2 negative)( ATCC, Rockville, MD). Cells were cultured in a humidified atmosphere with 5% carbon dioxide at 37 °C (BINDER, Tuttlingen, Germany) in Dulbecco modified Eagle medium (Biowest, Nuaillé, France) supplemented with 10% fetal bovine serum (Biowest, France) and 1% penicillin/streptomycin 10,000 U/mL (Merck Millipore, Darmstadt, Germany).

The MCF-7 (ER positive, PR positive, HER2 negative) and the MDA-MB-468 (ER negative, PR negative, HER2 negative) cell lines were obtained from American Type Culture Collection (ATCC). Cells were cultured in a humidified atmosphere with 5% carbon dioxide in air at 37 °C. Both cell lines were cultured in Dulbecco modified Eagle medium (Biowest, France) supplemented with 10% foetal bovine serum (Biowest, France) and 1% penicillin/streptomycin 10,000 U/ml (Merck Millipore, Germany). The MCF-7 cells were additionally supplemented with 0.01 mg/ml insulin (Bioton, Poland).

Induction of vi-VIM after HBV infection was analyzed in human HepG2 hepatoma cells, while protein presence was assessed in the liver of HBV-uninfected and -infected patients. HepG2 cells were purchased from the Korean Cell Line Bank (Seoul, Republic of Korea) and cultured in Dulbecco Modified Eagle Medium (Biowest, Riverside, MO, USA) until confluence. HBV was collected from the supernatant of HepG2.2.15 cells (Korean Cell Line Bank) and enriched for viral particles with the PEG Virus Precipitation kit (BioVision; Milpitas, CA, USA) by following the manufacturer’s protocol. HepG2 cells were then infected with increasing doses of precipitated HBV (i.e., 50, 150, and 300 μL) and harvested 2-, 4-, 8-, or 12-h post-infection (pi), while noninfected cells served as a control. Cells were subjected to Western blot analysis or immunocytochemistry. A human liver tissue array was purchased from US Biomax (Derwood, MD, USA; cat. no. LV1601) and subjected to immunohistochemistry (IHC) or immunofluorescence staining. The array contains doublets of 80 liver tissues. HBV-uninfected samples include noncancerous liver tissues (35 samples), while HBV-infected samples consist of liver tissues with CHB (13 samples) or cirrhosis (32 samples).

The A549 cells are adenocarcinomic human alveolar basal epithelial cells and have served as models of human alveolar type 2 pulmonary epithelium in investigations into the metabolic processing of lung tissue and possible mechanisms of drug delivery to such tissue. The A549 cells were purchased from the Korean Cell Line Bank. The A549 cells were cultured in basal medium and grown in 100 mm plates (Corning Inc., Corning, NY, USA) at 37 °C in 5% CO₂. The basal medium consisted of DMEM (Biowest, USA) supplemented with certified fetal bovine serum (FBS; Biowest Nuailléa, France), penicillin (100 U/mL) and streptomycin (100 µg/mL; Biowest). To confirm the effect of DEX in the A549 cell line, A549 cells were transferred to DMEM without phenol red and with 5% charcoal/dextran-treated FBS instead of normal FBS. Adherent cells were grown for 1 day, and, on the next day (2 day), the A549 cells were treated with 10⁻⁸ M DEX for 24 h. Some of the 10⁻⁸ M DEX-treated groups were also treated with 10⁻⁶ M of RU486, a DEX antagonist.