Roswell park memorial institute (rpmi)

All chemicals, unless otherwise stated, were purchased from Sigma (St. Louis, MO, USA). Recombinant TRAIL (rTRAIL) was from R&D Systems (Minneapolis, MN, USA). Docetaxel, AKTi (MK-2206) and PI3Ki (LY294002) were from Stratech (Stratech Scientific, Ely, UK).
The colorectal carcinoma cell lines Colo205 (obtained from Dr. Eva Szegezdi) and HT-29 (ATCC, Manassas, VA, USA) were cultured in RPMI (Lonza, Basel, Switzerland) and in McCoy’s 5A (Lonza), respectively. HEK293 cells were grown in DMEM (Lonza) and CHO cells (ATCC) in Ham’s F12. The prostate cancer cell lines, LNCaP (ATCC), C4-2B (obtained from Dr. Wafa Al-Jamal) and PC3 (ATCC), were cultured in RPMI medium (Lonza). All media were supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific, Waltham, MA, USA), 100 U/mL penicillin and 100 μg/mL streptomycin.
Human bone marrow derived MSCs were from Lonza, human adipose derived stem cells were from Amsbio (Cambridge, MA, USA) and human umbilical cord derived MSCs were from Promocell (Heidelberg, Germany). The different human MSCs were cultured in StemMACS MSC Expansion Medium (Miltenyi Biotec, Bergisch Gladbach, Germany). Murine MSCs were isolated and cultured as previously described [75 (link)]. In experiments where it was stated that MSCs were used, but not further defined, human bone marrow derived MSCs were used.

Thawed PBMC (≥80% viable) were plated in a 96-well plate after 4 h of resting (EuroClone) at a concentration of 1x10 [6 (link)] PBMC/well in complete RPMI medium [10% FBS (Lonza-BioWhittaker) in RPMI (Lonza-BioWhittaker)] with single/pools of HIV-1 derived peptides (2 μM) in the presence of a mixture of co-stimulatory anti-CD28 and anti-CD49d Ab (1.3 μg/ml each, Becton Dickinson). Cells were then treated and stained as previously described [13 (link)]. Detailed methods are reported in the Additional file 1 section.

C4-2 human prostate cells were grown in RPMI (Lonza, Alpharetta, GA) supplemented with 10% fetal bovine serum (Atlanta Biologicals, Flowery Branch, GA) and 1 × penicillin-streptomycin solution (Mediatech, Manassas, VA) at 37°C in a humidified incubator with 5% CO₂. MSKE, which is composed mainly of anthocyanins, was prepared as previously described [7 (link)]. The protease inhibitor cocktail was purchased from Roche Molecular Biochemicals (Indianapolis, IN) and used according to the manufacturer’s instructions. Chloroquine (autophagy inhibitor) was purchased from Sigma Aldridge.(St. Louis, MO). Annexin V/cell death apoptosis kit was purchased from Thermo Fisher Scientific (Waltham, MA).

Anticancer effect of ALF, Se NPs and ALF-Se NPs was assayed using three human cancer cell lines, including breast cancer cell line (MCF-7), liver cancer cell line (HepG-2) and colon cancer cells (Caco-2). All cell lines were obtained from the American Type Culture Collection (ATCC, USA). MCF7 and HepG2 were maintained in RPMI (Lonza, USA) while Caco2 cell line was cultured in DMEM (Lonza, USA), both media were supplemented with 10% FBS. All cancer cells (5 × 10³ cells/well) were seeded in sterile 96-well plates. After 24 h, serial concentrations of ALF (62.5, 125, 150, 500, 1000 and 2000 ug/ml), Se NPs and ALF-Se NPs (31.25, 62.5, 125, 150, 500 and 1000 ug/ml) as compared to 5-FU (standard reference drug) were incubated with four cancer cell lines for 72 h at 37 °C in 5% CO₂ incubator. MTT method was done as described above. The half maximal inhibitory concentration (IC₅₀) values were calculated using the Graphpad Instat software. Furthermore, cellular morphological changes before and after treatment with ALF, Se NPs and ALF-Se NPs were investigated using phase contrast inverted microscope with a digital camera (Olympus, Japan). Importantly, selectivity index (SI) that defined as the ratio of the IC₅₀ on normal cells versus tumor cells of ALF with or without Se NPs was estimated as IC₅₀-N/IC₅₀^{75 (link)}.