Streptomycin

NK-92 cells were cultured in X-VIVO 10 medium (Lonza, Cologne, Germany) supplemented with 5% human plasma (German Red Cross, Dresden) and 500 IU/mL IL-2 (Proleukin S; Novartis Pharmaceuticals, Horsham, UK). The 3T3 cell line used for the production of target modules and HEK 293T human embryonic kidney cells were bought from American Type Culture Collection (ATCC, Manassas, VA, USA). JF neuroblastoma cells (kindly provided by Prof. Malcolm K. Brenner, Houston, TX, USA), FM3, MZ-Mel 2, NW-Mel 450 and Panc-89 cells were transduced with a lentiviral vector for stable expression of firefly luciferase, yielding JF Luc, FM3 Luc, MZ-Mel 2 Luc, NW-Mel 450 Luc and Panc-89 Luc cells as described previously^{33 (link),62 (link),}. JF, FM3 and Panc-89 cells were cultured in RPMI 1640 medium supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin, 1% non-essential amino acids, 1 mM sodium pyruvate, 2 mM N-acetyl-L-alanyl-L-glutamine and 10% FCS (Biochrom, Berlin). 3T3 and HEK 293T, MZ-Mel 2, NW-Mel 450 cell lines were cultured in DMEM medium supplemented with 100 μg/ml streptomycin and 100 U/ml penicillin, 1% non-essential amino acids and 10% FCS (Biochrom, Berlin). All cells were kept at 37 °C with 5% CO₂, and cultivated twice every week when they were around 90% confluent.

For co-transfection experiments the human embryonic kidney cell line HEK293AD (Stratagene, La Jolla, CA, USA) was grown in DMEM supplemented with 10% fetal calf serum (FCS) and 100 U/mL penicillin/100 μg/mL streptomycin (Biochrom, Berlin, Germany) at 37 °C and 5% CO₂ in a humidified incubator. Trypsin (0.05%)-EDTA (0.02%) (Biochrom, Berlin, Germany) was used for detachment of cells, and cells were pelleted by centrifugation at 350× g for 5 min. For transient transfection of DNA the jetPEI reagent (Polyplus-transfection SA, Illkirch, France) was used according to the manufacturer’s instructions. The transfected DNA amounts varied in the different experiments: Section 2.1: 1.5 μg target molecule + 1.5 μg RTM were transfected into HEK293 cells cultivated in a 6 well plate; Section 2.2 and Section 2.4: 3 μg COL7A1-MG + 3 μg dRTM were co-transfected into HEK293 cells and 5 μg of dRTM were transfected into the stably COL7A1-MG-expressing cell line cultivated in 60 mm plates. The RDEB patient keratinocytes carry a homozygous mutation (6527insC) in exon 80 of COL7A1 (RDEB-TA4) [26 (link)]. This cell line was cultivated in SFM medium (Life technologies, Carlsbad, CA, USA) including the provided supplements and 100 U/mL penicillin/100 μg/mL streptomycin (Biochrom) at 37 °C and 5% CO₂ in a humidified incubator.

Doxorubicin, superoxide dismutase (SOD), iron chloride (FeCl₃), nitro blue tetrazolium (NBT), ascorbic acid (AA), catalase (CAT) from bovine liver, acetaminophen (APAP), 2,2′-Diphenyl-1-picrylhydrazyl (DPPH), n-alkanes (C6–C30), thiobarbituric acid (TBA), trichloroacetic acid (TCA), ethylenediaminetetra acetic acid (EDTA), nicotinamide-adenine dinucleotide phosphate (NADPH), butylated hydroxytoluene, gallic acid, sodium carboxymethyl cellulose (CMC), phenazine methosulfate, Folin–Ciocalteu reagent, reduced glutathione (GSH), and 1,2-dithio-bis nitro benzoic acid (DTNB) were purchased from Sigma Co. (St. Louis, MO, USA). Dimethyl sulfoxide (DMSO) was purchased from Merck (Merck, Darmstadt, Germany). Streptomycin, Dulbecco’s modified Eagle’s medium (DMEM), and Streptomycin were purchased from Biochrom (Biochrome AG, Berlin, Germany, A 321-44). Fetal bovine serum was purchased from Sigma (Sigma, Darmstadt, Germany). Potassium phosphate monobasic, sodium pyrophosphate dibasic, sodium carbonate (Na₂CO₃), disodium hydrogen phosphate (Na₂HPO₄), hydrogen peroxide (H₂O₂), potassium ferricyanide (K₃Fe(CN)₆), sodium sulphate anhydrous (Na₂SO₄), acetic acid (ACA), and n-butanol (99.8%) were of analytical grade and purchased from Merck (Nottingham, UK).

The GRPR positive human prostate adenocarcinoma cell line PC-3 (Reile et al. 1994 (link)) was purchased from American Type Culture Collection (ATCC via LGC Standards AB, Borås, Sweden) and grown in Roswell Park Memorial Institute-1640 medium (RPMI-1640, Sigma Aldrich, Saint Louis, MO, USA) containing 1% (v/v) L-glutamine and supplemented with 10% (v/v) fetal bovine serum (FBS, Sigma Aldrich, St. Louis, MO, USA) as well as 1% (v/v) penicillin/streptomycin solution (PEST; 10 000 U/mL penicillin; 10 000 µg/mL streptomycin, Biochrom, Berlin, Germany). Cells were incubated in a 5% CO₂ humidified atmosphere at 37 °C in 75 cm² cell culture flasks with vent caps (Corning®, Corning, NY, USA) in a Sanyo MCO-19AIC incubator (SANYO Electric Co., Ltd, Osaka City, Osaka, Japan). For sub-culturing a 0.25% trypsin–EDTA solution (Sigma Aldrich, St. Louis, MO, USA) was used.

Normal human dermal fibroblasts (NHDF) were isolated from skin following plastic surgery interventions after approval by the local ethics committee of the University Hospital in Pilsen, E. Benese 13, 305 99 Pilsen, Czech Republic, decision of November 5th, 2015. The guidelines in the Declaration of Helsinki were followed. All donors gave written informed consent before intervention. The samples were washed by Hank’s balanced salt solution (HBSS) (Merck KGaA, Darmstadt, Germany) containing penicillin (100 U/ml)/streptomycin (0.1 mg/ml) (Biochrom, Cambridge, UK) and gentamicin (50 μg/ml) (Biochrom). The samples were cut and digested overnight at 37 °C in HBSS containing collagenase type I (100 U/ml, Merck KGaA). On the following day, the suspension was shaken intensively and filtered through 100 µm nylon cell strainer (Falcon™, Thermo Fisher Scientific, Waltham, Massachusetts). The cell suspension was then transferred to a cultivation flask (TPP) containing low glucose Dulbecco’s Modified Eagle’s Medium (DMEM) (Merck KGaA), 10% heat-inactivated fetal bovine serum (FBS) (Merck KGaA), penicillin (100 U/ml)/streptomycin (0.1 mg/ml) (Biochrom), 0.5% L-glutamin (Biosera, Nuaille, France) and 1.0% non-essential amino acids (Biosera). The NHDF were cultured at 37 °C, 5% CO₂ up to 80% confluence and then passaged. Only those NHDF from the 2th–5th passages were used in the experiments.

The neuroblastoma cell line JF was kindly provided by Malcolm K. Brenner, Houston, USA. The Ewing’s sarcoma cell line TC-71 was obtained from DSMZ (Braunschweig, Germany). The prostate cancer cell line PC3 as well as the CHO cell line were purchased from American Type Culture Collection. For the bioluminescence based killing assay and the in vivo analysis JF and TC-71 cells were transduced to express the gene encoding firefly luciferase (JF Luc, TC-71 luc). Transduction was performed using a lentiviral packaging system as described previously [39 (link)]. PC3 cells expressing firefly luciferase were described previously [36 (link)]. All these cell lines were cultured in RPMI 1640 medium completed with 10% FCS, 100 U/ml penicillin and 100 µg/ml streptomycin, 2 mM N-acetyl-L-alanyl-L-glutamine, 1% non-essential amino acids and 1 mM sodium pyruvate (Biochrom). Human Embryonic Kidney cells HEK293T (ATCC CRL-11268) used for production of the lentiviral particles were cultured in DMEM medium supplemented with 10% FCS, 100 U/ml penicillin and 100 μg/ml streptomycin, 1% non-essential amino acids (Biochrom). Cells were maintained at 37°C in a humidified atmosphere of 5% CO₂.