Penicillin g

The human pancreatic cancer cell lines PANC-1, MIA PaCa-2, and SW1990 were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA); AsPC-1, BxPC-3, Capan-2, and Panc 03.27 cells were purchased from the Cell Repository of the Chinese Academy of Sciences (Shanghai, China). The immortalized HPDE cell line was obtained from the Beijing North Carolina Chuanglian Biotechnology Research Institute (Beijing, China). Capan-2, MIA PaCa-2, and PANC − 1 cells were grown in Dulbecco Modified Eagle Medium (Gibco, Carlsbad, CA, USA) supplemented with 10% FBS (Gibco), 100 U/mL penicillin G, and 100 mg/mL streptomycin (Sigma-Aldrich, St. Louis, MO, USA). AsPC-1, BxPC-3, Panc 03.27, and HPDE were grown in 1640 medium (Gibco) supplemented with 10% FBS (Gibco), 100 U/mL penicillin G, and 100 mg/mL streptomycin (Sigma-Aldrich). All cells were grown at 37 °C in a humidified 5% CO₂ incubator. The reagents and antibodies used in this study are listed in the Additional file 10: Table S5. Reagents and antibodies.

Cell lines, namely LNCaP, PC-3, T47-D and NHDF were obtained from American Type Culture Collection (ATCC; Manassas, VA, USA) and cultured in 75 cm² culture flasks at 37 °C in a humidified air incubator with 5% CO₂. LNCaP, PC-3 and T47-D cells, which were used in passages 20th to 27th, 25th to 29th and 10th to 15th, respectively, were cultured in RPMI 1640 medium (Sigma-Aldrich, Inc., St. Louis, MO, USA) with 10% fetal bovine serum (FBS; Sigma-Aldrich, Inc. St. Louis, MO, USA) and 1% of the antibiotic mixture of 10,000 IU/mL penicillin G and 100 mg/mL of streptomycin (Sp, Sigma-Aldrich, Inc. St. Louis, MO, USA). Finally, NHDF cells (Normal Human Dermal Fibroblasts) were cultured in RPMI 1640 medium supplemented with 10% FBS, 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate and 1% of an antibiotic/antimycotic mixture (10,000 U/mL penicillin G, 100 mg/mL streptomycin and 25 µg/mL amphotericin B) (Ab; Sigma-Aldrich, Inc. St. Louis, MO, USA), and these cells were used in passages 10 to 12. For all cell types, the medium was renewed every 2–3 days until cells reach nearly the confluence state. Then, they were detached gently by trypsinization (trypsin-EDTA solution: 0.125 g/L of trypsin and 0.02 g/L of EDTA).

Oral and cloacal swabs obtained from each bird were placed in separate tubes containing 1.5 ml of brain-heart infusion broth (BHI) with antibiotics (2000 U/ml penicillin G, 200 μg/ml gentamicin sulfate, and 4 μg/ml amphotericin B; Sigma Chemical Co., St. Louis, MO). Swab sample tubes were centrifuged at 1000 × g for 20 min and the supernatant removed for virus isolation and titration in eggs, according to standard procedures [5 , 43 ].
Harvested spleens were homogenized with a Stomacher in a 10 % w/v solution with PBS supplemented with antibiotics (2000 U/ml penicillin G, 200 μg/ml gentamicin sulfate, and 4 μg/ml amphotericin B [Sigma Chemical Co., St. Louis, MO]). For each sample, virus isolation was performed by inoculating undiluted homogenized samples into three embryonated eggs (200 μL per egg). Virus titer in positive eggs was assessed in embryonated eggs, as per standard procedures [5 , 43 ].
Whole blood from chickens was harvested into EDTA-containing tubes and 200 μL were inoculated into three SPF embryonated eggs per sample for virus isolation (VI). Virus titers from VI-positive samples were subsequently assayed in embryonated eggs [5 , 43 ].

Breast (MCF-7, T47-D), prostatic (LNCaP) and colon (Caco-2) cancer cells, as well as normal human dermal fibroblasts (NHDF), were obtained from American Type Culture Collection (ATCC; Manassas, VA, USA). The hepatic (HepaRG) cell line was acquired to Life Technologies-Invitrogen™ (through Alfagene, Portugal). They were cultured in 75 cm² culture flasks at 37 °C in a humidified air incubator with 5% CO₂. MCF-7 cells were maintained with high-glucose Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich, St Louis, MO, USA) and 1% antibiotic/antimycotic mixture (10,000 units/mL penicillin G, 100 mg/mL streptomycin and 25 μg/mL amphotericin B) (Sigma-Aldrich, St Louis, MO, USA). High glucose DMEM supplemented with 10% FBS and 1% of the antibiotic mixture of 10,000 units/mL penicillin G and 100 mg/mL of streptomycin (Sigma-Aldrich, St Louis, MO, USA) was used for Caco-2 cells. LNCaP and T47-D cells were cultured in RPMI 1640 medium with 10% FBS and 1% antibiotic mixture. NHDF cells grew in RPMI 1640 medium supplemented with 10% FBS, 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate and 1% antibiotic/antimycotic. Lastly, hepatic cells were seeded in Williams’ E medium supplemented with 10% FBS, 1% antibiotic mixture, 5 µg/mL insulin, and 5 × 10⁻⁵ M hydrocortisone hemisuccinate (Sigma–Aldrich, St Louis, MO, USA).

Quantification of penicillin G in culture supernatant was performed by LC-MS as developed previously, using an Agilent 1290 LC and 6550 quadropole time-of-flight mass spectrometer with electrospray ionisation and an Agilent Zorbax Extend C-18, 2.1 × 50 mm, 1.8 μm LC column^{19 (link)}. penicillin G (Sigma Aldrich) standards of concentration 0 ng ml⁻¹, 10 ng ml⁻¹, 100 ng ml⁻¹, 1 μg ml⁻¹ and 10 μg ml⁻¹ were used to construct a curve of best fit that was then used to quantify penicillin G in culture supernatant samples. Samples were prepared by growing cells to an OD₆₀₀ of 0.6, in 96-well format, centrifuging and removing the supernatant, which was used as the sample.

Primary fibroblasts (passage 4) were seeded at 4 × 10³ cells/cm² and cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FB Essence serum (FBe; Seradigm, Salt Lake City, UT, USA), 100 UI/mL penicillin G (Sigma, Oakville, ON, Canada), and 25 μg/mL gentamicin (Gemini, West Sacramento, CA, USA). Primary keratinocytes (passage 1) were seeded at 4 × 10³ cells/cm² on a feeder layer of irradiated 3T3 murine fibroblasts and cultured in a combination of DMEM with Ham’s F12 in a proportion of 3:1 (DMEMH), supplemented with 5% Fetal Clone II serum (Hyclone, Scarborough, ON, Canada), 5 μg/mL insulin (Sigma, St. Louis, MO, USA), 0.4 μg/mL hydrocortisone (Galenova, Saint-Hyacinthe, QC, Canada), 0.212 μg/mL isoproterenol hydrochloride (Sandoz Canada, Boucherville, QC, Canada), 10 ng/mL human epidermal growth factor (EGF; Austral Biological, San Ramon, CA, USA), 100 UI/mL penicillin G (Sigma, Oakville, ON, Canada) and 25 μg/mL gentamicin (Gemini, West Sacramento, CA, USA). Cell cultures were incubated at 37 °C in an 8% carbon dioxide (CO₂) atmosphere. Cell culture media were changed every two days, for a total of three times per week.