Penicillin streptomycin amphotericin b

Human OS cell lines U2-OS and 143B (ATCC, Gaithersburg, MD, USA) were cultured according to the manufacturer’s instructions. U2-OS cells were cultured in low glucose Dulbecco’s Modified Eagle Medium (Low DMEM) supplemented with 10% FBS, 1% penicillin–streptomycin–amphotericin B, and 1% glutamine (Biological Industries Ltd.). 143B cells were cultured in Minimum Essential Medium Eagle (MEM-Eagle) with 0.015 mg/mL 5-bromo-2′deoxyuridine (SIGMA-ALDRICH, Burlington, MA, USA) supplemented with 10% FBS, 1% penicillin–streptomycin–amphotericin B, and 1% glutamine (Biological Industries Ltd.). Cells were cultured at 37 °C in a humidified atmosphere of 95% air/5% CO₂. Cells were fed twice a week, and when reachinhg 80–90% confluence, they were split by trypsinization, using a solution containing 0.5% trypsin in 0.25% ethylenediaminetetraacetic acid (EDTA) (Biological Industries Ltd.).

HEK293T cells with passage numbers smaller than 22, were maintained grown in high glucose DMEM medium with stable L-glutamine (Lonza, Verviers, Belgium), supplemented with 10% fetal bovine serum (Gibco, Thermo Fisher Scientific) and 1% penicillin/streptomycin/amphotericin B (Biological Industries, Beit-Haemek, Israel), under standard 80%–90% humidity, 5% CO2 cell culture environment. CaPO4 precipitation method was utilized for transfections. TNFα stimulations were performed following 16 h serum starvation and 1-h pretreatment with 100 µM sodium orthovanadate (Na3VO4).

Extralobar PAs were dissected carefully and prepared for tissue culture. Ex vivo explant cultures were executed according to our previous reports. The vascular endothelium was removed using a sterile cotton swab with mild rubbing. Then, we carefully removed the tunica adventitia and the superficial part of the tunica media. The remaining pieces were cut into small pieces and then transferred to culture flasks for further cultivation. The ex vivo explants were incubated in Dulbecco’s Modified Eagle Medium (DMEM; Gibco Laboratories, Gaithersburg, MD, USA) with 10% fetal bovine serum and 1% penicillin-streptomycin amphotericin B (Biological Industries, Kibbutz Beit Haemek, Israel) to promote cell growth. The incubator was set to 37 °C with a humidified 5% CO₂ environment. PASMCs began to proliferate from explants after culture for 7 days. The growth of PASMCs was arrested by substituting the media with FBS-free DMEM. Then, the cells were incubated in the absence and presence of test agents under normoxia (20% O₂) or hypoxia (1% O₂) for 24 h at 37 °C [21 (link)]. No more than five passages of primary cultures were used in the subsequent experiments. The immunofluorescent staining of α-actin was used to confirm the purity of PASMCs.

Human HCC cell lines Huh-7, HepG2, HA59T, HA22T, HCC36, and Mahlavu and sorafenib-resistant cells (Huh-7/SR) were grown in Dulbecco’s modified Eagle medium (Corning, USA) supplemented with 10% fetal bovine serum and 100 U/mL penicillin–streptomycin–amphotericin B (Biological Industries) and were incubated at 37° C in a humidified atmosphere containing 5% CO₂. To induce sorafenib resistance, Huh-7 cells were treated with 0.25 μM sorafenib (BAY 43-9006, Selleck Chemicals, USA), and the dose was gradually increased every week for 6 months. The resistant cells were maintained at a final dose of 10 μM.

Copper(II) chloride dehydrate (CuCl₂·2H₂O), hydrochloric acid (HCl, 37%), nitric acid (HNO₃, 65%), and Dulbecco’s modified Eagle’s medium-high glucose (DMEM/HG) were purchased from Thermo Fisher Scientific (Massachusetts, USA). Iron(II) chloride anhydrous (FeCl₂, 99.5%), methylene blue (C₁₆H₁₈ClN₃) and thiazolyl blue tetrazolium bromide (MTT) were from Alfa Aesar (Massachusetts, USA). Hydrazine hydrate (N₂H₄·H₂O) and sodium pyruvate (C₃H₃NaO₃S·xH₂O) were from Acros Organics (Morris Plains, NJ, USA). poly(styrene-alt-maleic acid) sodium salt solution (PSMA), hydrogen peroxide (H₂O₂), 2′,7′-dichlorofluorescein diacetate (DCFH-DA), sodium bicarbonate (NaHCO₃), trypan blue (0.4%), imidazole, N,N-dimethyl-4-nitrosoaniline (RNO), N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), and folic acid (C₁₉H₁₉N₇O₆) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum (FBS), trypsin-ethylenediaminetetraacetic acid(trypsin-EDTA, 0.25%) and antibiotic-antimycotic (penicillin/streptomycin/amphotericin B) were from Biological Industries (Cromwell, CT, USA). Dimethyl sulfoxide was from Scharlau (Barcelona, Spain).