Mem medium

The HapT1 cancer cell line was commercially procured from Sigma, and human PSCs (hu-PSCs) were procured from Sciencell Research Laboratories. Both the cell lines were propagated and frozen immediately after receipt. The frozen stocks were revived and used within three months. HapT1 cells were maintained in 10% FBS that contained MEM medium (Pan-Biotech) supplemented with 1x NEAA (Life Technologies). Human PSCs were maintained in 2% FBS containing Stellate Cell Medium (SteCM; Sciencell) supplemented with growth factors. With the approval of Institutional Animal Ethics Committee (Institute of Life Sciences, Bhubaneswar, India), rat and hamster PSCs were isolated from normal pancreas. The PSCs were confirmed to have the typical stellate cell morphology and were positive for α-SMA (a marker for activated PSCs). All of the established cell lines were used between 3-6 passages. Rat PSCs were maintained in 10% FBS containing Iscove's Modified Dulbecco's Medium (IMDM; Pan-Biotech), and hamster PSCs were maintained in 5% hamster serum containing IMDM medium (Pan-Biotech). To check cell viability in vitro for HapT1 and PSCs, the MTT assay, crystal violet staining, and colony formation assay were performed as reported earlier [44 (link), 45 (link)]. To determine the role of ROS-induced cell death, cells were pre-treated with 1 mM NAC for 12 hours before adding DSF or DSF+Cu.

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).

Human lung carcinoma cell line A549 and CCD-18Co colon fibroblasts were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA). The A549 cells were cultured in a complete RPMI-1640 medium (Sigma-Aldrich, St. Louis, MO, USA), and CCD-18Co cells were cultured in an MEM medium (PAN-Biotech GmbH, Aidenbach, Germany). The media were supplemented with 10% fetal bovine serum (FBS; Biosera, Nuaille, France) and antibiotics (1% Antibiotic-Antimycotic 100× and 50 × 10⁻³ g l⁻¹ gentamicin; Biosera) at 37 °C, 95% humidity, and 5% CO₂.
Prior to the selected treatments, cells were seeded on 12-well μ-Chamber slides (ibidi GmbH, Martinsried, Germany) and 6 and/or 96-well plates (TPP, Trasadingen, Switzerland) and left to settle for 24 h. The biscoumarin derivative solutions (at concentrations ranging from 10–100 µM) were then added to cells for 24 or 48 h, and analysis was subsequently performed.