Mem medium

The protective effects of the isolated compounds were evaluated on the defense effect of SH-SY5Y human neuroblastoma cells (Chinese Academy of Science Committee Type Culture Collection Cell, Bank, Shanghai, China) against H₂O₂-induced cytotoxicity. The SH-SY5Y cells were maintained in MEM medium (Sigma-Aldrich, St. Louis, MO, USA) in a humid atmosphere of 5% CO₂ and 95% air at 37 °C, then the cells were treated with 350 μM H₂O₂ for 24 h. The conventional MTT colorimetric assay was applied to measure the neuroblastoma cell viability, and we used the cells without pre-treatment as the control. The SH-HY5Y cells were plated into 96-well plates at a density of 1 × 10⁴ per well and incubated for 24 h. The experimental groups were maintained in MEM supplemented with 1 μM of the obtained depsides 1–12 at the same condition of the control groups for 15 min, then they were exposed to 350 μM H₂O₂ for 24 h. Each group was further incubated with MTT (Sigma-Alarich, St. Louis, MO, USA) for another 4 h, the supernatant was removed after that, the crystals were dissolved in DMSO. The optical density of each well was evaluated by a microplate reader (BIO-RAD Model 680, Laboratories, Hercules, CA, USA) at 492 nm.

Rainbow trout RBCs were obtained and purified as previously described (4 (link), 7 (link)). Briefly, RBCs extracted from the caudal vein were purified by 2 successive Ficoll density gradient centrifugations (7,206 g, Ficoll 1.007; Sigma-Aldrich, Madrid, Spain). Ficoll-purified RBCs were maintained in 25 cm² flasks (Nunc Roskilde, Denmark) with RPMI-1640 medium (Dutch modification) (Gibco, Thermo Fisher Scientific, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS) gamma irradiated (Cultek, Madrid, Spain), 1 mM pyruvate (Gibco), 2 mM L-glutamine (Gibco), 50 μg/mL gentamicin (Gibco), 2 μg/mL fungizone (Gibco), 100 U/mL penicillin (Sigma-Aldrich), and 100 μg/mL streptomycin (Sigma-Aldrich) at 14°C for 24 h prior to experimentation.
The RTG-2 (rainbow trout gonad-2) cell line was purchased from the American Type Culture Collection (ATCC 50643) and maintained at 21°C in MEM medium (Sigma-Aldrich) containing 10% FBS, 1 mM pyruvate, 2 mM L-glutamine, 50 μg/mL gentamicin, and 2 μg/mL fungizone.
VHSV strain 07.71 (20 ) was purchased from the American Type Culture Collection (ATCC VR-1388) and cultured in fathead minnow epithelioma papulosum cyprini (EPC) (21 (link)) cells at 14°C as previously described (22 ).

5×10⁴ cells MC3T3-E1 were seeded on non-corroded Mg10Gd, Pure Mg and Mg2Ag. After incubation for 2, 4, 8 and 12 days at 37°C, 5% CO₂ in Minimum Essential Medium Eagle(MEM) medium (Sigma-Aldrich) supplemented with 10% FBS (Sigma-Aldrich), samples were washed twice with PBS and fixed with 4% formaldehyde (Merck) for 10 minutes at RT. After washing twice with PBS, the cells were permeabilized with 0.3% Triton X100 (Sigma-Aldrich) in PBS for 5 minutes at RT. The cells were washed three times in PBS. Blocking solution prepared in 0.3% Triton X100 contained 1% Bovine serum albumin (Sigma-Aldrich) and 5% horse serum (Sigma-Aldrich). A rabbit anti-Collagen 1 polyclonal (Catalogue number ab21286; Abcam, Cambridge, UK) was used as a primary antibody at a dilution of 1:100 for 4 hours in RT. Highly cross adsorbed anti-rabbit IgG (H+L) (Sigma-Aldrich) was used as secondary antibody at a 1:200 dilution. Pictures were taken using a laser scanning microscope (Zeiss, LSM 510).

Human cancer cells (Hep G2) ATCC® 59195™ were used in the study. The cells were cultured in standard conditions (37 °C, 5% CO₂), in MEM medium (Sigma-Aldrich), supplemented with 10% FBS (Sigma-Aldrich) and antibiotics (Sigma-Aldrich).

The mT4-2D cell line was a kind gift from Professor David Tuveson (CSHL) and was maintained in complete Dulbecco′s Modified Eagle′s Medium (DMEM) (Sigma, St. Louis, MO, USA) containing 10% foetal bovine serum (FBS) (Gibco, Waltham, MA, USA), 100 U/mL penicillin, 100 mg/mL streptomycin sulphate, and 2 mM L-glutamine [42 (link)]. The KPC (KrasLSL.G12D/+; p53R172H/+; PdxCretg/+) primary cell line, which we established as previously described [29 (link)], was cultured in standard cell culture vessels with DMEM supplemented with 10% FBS and added 1% Penicillin/Streptomycin. All other cancer cell lines were acquired from ATCC. HPAF-II was cultured in MEM medium (Sigma); AsPC-1, A2780, PC3, and 4T1 were cultured in RPMI 1640 medium (Sigma).
All the culture media, except for the cancer stem-like cell medium, were supplemented with 1% penicillin-streptomycin-glutamine (PSG) and 10% of FBS. Cancer stem-like cells were cultured in DMEM/F-12 medium (Sigma) supplemented as previously described and treated according to the protocol [29 (link)]. Cells were maintained in a humidified chamber (Nuaire DH auto flow CO₂ air-jacketed incubator) at 37 °C with 5% CO₂.