Minimal essential medium

Adult T. canis worms were collected from feces of dewormed dogs treated in veterinary clinics in Warsaw. Eggs were obtained from dissected female worms and incubated in 0.1 N H₂SO₄. Fully embryonated and infective eggs were hatched as described by Oaks and Kayes (1979 (link)) and maintained in vitro in Minimal Essential Medium (Sigma-Aldrich) supplemented with penicillin (100 U/ml), streptomycin (100 μg/ml), and amphotericin B (2.5 μg/ml) at 37 °C, 5 % CO₂. Culture medium was replaced every 3 days, and the spent medium was collected, concentrated, and dialysed against sterile phosphate-buffered saline (PBS) using Amicon Ultra Centrifugal Units (Millipore). TES solution was filtered through 0.22-μm filter, and antigen concentration was determined using BCA Protein Assay (Thermo Scientific).

MDCK II (Madin-Darby canine kidney) and HEK293T (Human embryonic kidney) cells were cultured in MEM (Minimal Essential Medium; Sigma-Aldrich) or DMEM (Dulbeccós Modified Eaglés Medium; Sigma-Aldrich), respectively. Culture media were supplemented with 10% FBS (fetal bovine serum; Biochrom), 1% L-glutamine (Sigma-Aldrich) and 1% penicillin–streptomycin (Sigma-Aldrich). Cells were cultured at 37°C, 95% rH (relative humidity) and 5% CO₂.
The SC35 virus is an H7N7 HPAIV that is lethal in chicken and low pathogenic in mice and ferrets as described before [10 (link),11 (link)]. SC35F is the ferret-adapted variant of SC35 that is lethal in ferrets [11 (link)]. Recombinant SC35/SC35F SGR and SPM viruses were generated by reverse genetics using the pHW2000 plasmid system as described before [10 (link)]. The SC35 virus was grown in specific pathogen free 11 d embryonated chicken eggs (ValoBiomedia) as described before [10 (link)]. All recombinant SC35/SC35F SGR and SPM viruses, including the recombinant A/Netherlands/213/03 (H3N2) and A/Vietnam/11/94 (H5N1-HA_monobasic) viruses were propagated on MDCK II cells. The full SC35 gene sequence is deposited in the GenBank database (accession nos. DQ266094-DQ266101) [10 (link)].

The cell line used was NCTC-Clone 929, which was grown in Minimal Essential Medium (Sigma) supplemented with 10% heat-inactivated FBS, penicillin G (100 U/mL), and streptomycin (100 µg/mL). Cell cultures were maintained at 37 °C in a humidified 5% CO₂ atmosphere. The procedure for cell viability measurement was evaluated with resazurin by a colorimetric method. The cells were plated in 96-microtiter plates at 3 × 10⁴ cells per well in a 100 µL growth medium. The cells were grown overnight at 37 °C, 5% CO₂. Thereafter, the medium was removed and the compounds were added in a 200 µL medium for 24 h. After incubation, 20 µL of a 2 mM resazurin solution was added to each well. The plates were incubated for 3 h to allow optimal oxidation-reduction. The reduction of resazurin was determined by dual wavelength absorbance measurement at 490 and 595 nm. Background was subtracted. Each concentration was assayed three times. Medium and drug controls were used in each test as blanks [44 (link)].

To increase the stiffness of 3D collagen gels, we increased their concentration [34 (link), 35 ]. We utilized the high concentration collagen I solution (Corning 354249) and adjusted the pH using a modification of previously published protocols [37 (link)]. More specifically, we added the desired amount of collagen I so as to get a final collagen concentration of 0.5, 1.0 or 3.0 mg/ml, in a solution containing 10% 10x Minimal Essential Medium, 1% human insulin solution (Sigma-Aldrich), and distilled water. The pH was adjusted to 7.4 by adding 1N NaOH. Cancer cells were added to the collagen solution before it solidified at a concentration of 2.5 x 10⁵ cells/ml [37 (link)] and normal complete culture medium was added on top of the collagen gel containing cells 4h after its solidification. Cells were cultured in 3D collagen gels of 0.5, 1.0 or 3.0 mg/ml for 3 days and were then subjected to gene expression analysis at the mRNA and protein level as specified. In experiments involving RSU-1 silencing, siRNA transfection was performed in traditional 2D culture 1 day prior to embedding cells in the collagen gels. Cells were left to grow in the gels for two more days before being harvested and analyzed for gene expression.

Human fibroblasts (control: GM07492; HD: GM04281, 17/68 CAG in HTT gene, HD2: GM09197, 21/151 CAG in HTT gene) were obtained from Coriell Repository (Camden, NJ, USA). Fibroblasts were cultured in Minimal Essential Medium (Sigma–Aldrich, St. Louis, MO, USA) with 10% Fetal Bovine Serum (Sigma–Aldrich) supplemented with GlutaMAX (Life Technologies, Carlsbad, CA, USA), non-essential amino acids (Sigma–Aldrich), and Antibiotic Antimycotic Solution (Sigma–Aldrich) at 37°C. All cell cultures were checked for mycoplasma contamination.