Gentamycin solution

At UGA, adult female worms were pooled from three individual jirds and randomly distributed among 21 treatment groups (Table 1), ensuring each treatment included three independent replicates with 10 worms per replicate (3 × 10 worms per time point and drug concentration). Worms were washed in RPMI-1640 (BioWhittaker^® Classic Cell Culture Media, VWR, Mississauga, ON) supplemented with 1% v/v gentamycin (gentamycin solution, 10 mg/ml Sigma Aldrich Inc., St. Louis, MO, USA), prior to shipping on heat pads overnight to McGill in 15 mL of the same solution. Upon arrival, worms were allocated to individual culture plate wells containing 6 mL RPMI-1640 (Sigma-Aldrich Corp., St. Louis, MO, USA) supplemented with 10% v/v heat-inactivated fetal bovine serum (Sigma-Aldrich Corp., St. Louis, MO, USA), 5% penicillin/streptomycin (Sigma–Aldrich Corp., St. Louis, MO, USA) and 2% v/v gentamycin (Gibco, Thermo Fisher Scientific Inc., Grand Island, NY, USA) with or without drug. FLBZ (Epichem, Murdoch, WA, Australia) was prepared in 100% DMSO and diluted in media to a final concentration of 0.1% v/v DMSO; control media also contained 0.1% DMSO. Worms were incubated for 2 or 5 days at 37 °C and 5% CO₂, with daily media changes by replacing 3 mL of appropriate media.

In order to investigate the anti-proliferation activity of X. spinosum extract and fractions, T47D and MCF-7 (human epithelial breast cancer cells) were used, along with EMT6/P (mouse mammary cells) and Vero (kidney epithelial normal cells from African green monkey). The European Collection of Authenticated Cell Cultures (ECACC, Salisbury, UK) is the source of all the cell lines. The normal cell (Vero) was utilized to evaluate the toxicity of X. spinosum extract and fractions in vitro. EMT6/P cells were inoculated into the animals to induct breast tumors in mice. The tested cells were growing in a complete medium and incubated under specific conditions (5% CO₂ and 95% humidity). RPMI 1640 medium (PAN-biotech, Aidenbach, Germany) was used to culture the human breast cancer cells (T47D and MCF-7), while MEM medium (PAN-biotech, Aidenbach, Germany) was used to culture murine breast cancer cells (EMT6/P). For culturing normal cells (Vero), a DMEM medium was applied. The media was supplemented with 1% L-glutamine (Sigma, St. Louis, MO, USA), 10% fetal bovine serum (Gibco, UK), 1% penicillin-streptomycin (Sigma, St. Louis, MO, USA), and 0.1% gentamycin solution (Sigma, St. Louis, MO, USA).

N-phosphonacetyl-l-aspartate (PALA, NSC224131) was obtained from the National Cancer Institute (NCI)/Division of Cancer Treatment and Diagnosis (DCTD)/Developmental Therapeutics Program (DTP) Open Chemical Repository (http:dtp.cancer.gov). Aquaphor® and Neosporin® were purchased from Target (Cleveland Heights, OH). Gentamycin solution was purchased from Sigma (St. Louis, MO). Rabbit monoclonal anti-CAD antibody (EP711Y) was purchased from Novus Biologicals (Littleton, CO). Rabbit polyclonal anti-RICK (H-300) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit monoclonal anti-GAPDH (14C10) was purchased from Cell Signaling Technologies (Danvers, MA). Mouse monoclonal anti-α-tubulin (T5168) was purchased from Sigma. Antibodies against LL-37 (sc-166770) and HBD2 (sc-20798) were purchased from Santa Cruz Biotechnology. Anti-HBD3 antibody was purchased from Novus Biologicals (NB200-117). The plasmids, pcDNA3-HA-NOD1, pcDNA3-HA-NOD2, pBVIII-Luc, and pCMV-βgal were gifts of Gabriel Nuñez (University of Michigan) and have been previously described^{56 (link)}. MISSION short hairpin RNA (shRNA) constructs targeting CAD (NM_004341.3-6910s21c1), NOD2 (NM_022162.1-2959s1c1), RIP2 (NM_003821.5-2364s21c1), and non-targeting shRNA control (SHC002) were purchased from Sigma.

The antiproliferative activity of bran extracts was tested on four cell lines—three of them are human cancer cells and one is the normal cell line. MCF-7 and T47D are human epithelial breast cancer cell lines, while MDA-MB-231 is human hormone-independent breast cancer cells. EMT6/P and fibroblast are the mouse epithelial breast cancer cell line and human skin fibroblast cells, respectively. To achieve a successful cell culture, many factors were followed such as using completed medium and incubating cells in 5% CO₂ at 37 °C. Based on the type of the cells, the type of culturing medium varied. For MCF-7 and T47D, complete RPMI 1640 medium (PAN-biotech, Aidenbach, Germany) was used, while complete MEM medium (PAN-biotech, Aidenbach, Germany) was used for culturing EMT6/P. High-glucose DMEM medium (PAN-biotech, Aidenbach, Germany) was utilized for MDA-MB-231 and fibroblast cell lines. In this context, a complete culture medium was prepared through adding the following supplements with the required percentage of each type of tissue culture medium. The supplements are: 1% L-glutamine (Sigma, St. Louis, MO, USA), 10% fetal bovine serum (Gibco, Brough, UK), 1% penicillin-streptomycin (Sigma, St. Louis, MO, USA), 0.1% non-essential amino acids (Sigma, St. Louis, MO, USA) and 0.1% gentamycin solution (Sigma, St. Louis, MO, USA).

Tetraethyl orthosilicate (TEOS, Aldrich, ≥99%), (3-glycidyloxypropyl) trimethoxysilane (GPTMS, Fluka, ≥97%), cetyltrimethylammonium p-toluenesulfonate (CTATos, Sigma), triethanolamine (TEA, Aldrich, 98%), magnesium sulfate anhydrous (99.9%, Sigma), toluene anhydrous (Sigma), bi-distilled water obtained from a Millipore system (Milli-Q Academic A10). N(alpha),N(alpha)-bis(carboxymethyl)-L-lysine hydrate (NTA-lysine, Aldrich), sodium carbonate (Sigma), sodium bicarbonate (Sigma), nickel chloride hexahydrate (Riedel-de Haen), tris(hydroxymethyl)-aminomethane (TRIS, ≥99%, ROTH), acetic acid (99% – 100%, ROTH), thiazolyl blue tetrazolium bromide (MTT, ≥97.5%, Sigma), dimethyl sulfoxide molecular (DMSO, Applichem, biology grade), Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma), Dulbecco’s Phosphate Buffered Saline (PBS, Sigma), FBS Superior (Biochrom, S0615), Gentamycin solution (SERVA, 50 mg/ml), trypsin-EDTA solution (Sigma, T3924), Dulbecco’s Modified Eagle’s Medium – phenol red free (DMEM, Gibco), ethanol (EtOH, Aldrich, absolute).

Fish were anaesthetized by rapid chilling followed by cervical transection. The experiment was conducted in accordance with AVMA Guidelines for the Euthanasia of Animals. The fish skin tissue was then removed from the fish by scalpel and immediately immersed into cold L-15 medium (SIGMA, U.S.A.) supplemented with 10% fetal bovine serum, 2% gentamycin solution (SIGMA, U.S.A), 1× antibiotic–antimycotic (Biowest, U.S.A.).