Inactivated fetal bovine serum

MSCs were plated at density 1000 cells/cm² in 6-well plates and cultured in the platelet lysate supplemented basal culture medium with 100 IU/ml penicillin and streptomycin until 70–100% confluent. Adipogenic differentiation was induced for 3–4 days by an induction medium consisting of αMEM GlutaMax (Gibco, Life Technologies), 10% inactivated fetal bovine serum (Gibco, Life Technologies), 20 mM Hepes (Gibco, Life Technologies), 100 U/ml penicillin and streptomycin (Gibco, Life Technologies) supplemented with the induction cocktail of 0.1 mM indomethacin (Sigma), 44 μg/ml 3-isobutyl-methyl-xanthine (IBMX-22^*), 0.5 μg/ml insulin (Insulin-0.25^*) and 0.4 μg/ml dexamethasone (DM-200^*) (^*Preadipocyte Differentiation Medium Supplement Pack, PromoCell, Italy). Control cells were only maintained in induction medium without the induction cocktail. Differentiation was finalized by culturing the cells in a terminal differentiation medium consisting of induction medium supplemented with 0.1 mM indomethacin (Sigma), 0.5 μg/ml insulin (Insulin-0.25^*) and 3.0 μg/ml ciglitazone (Ciglitazone-1.5^*) (^*Preadipocyte Differentiation Medium Supplement Pack) for 2–4 weeks until visible lipid droplets could be observed. The cells were fixed with 4% PFA and were stained using Sudan III.

Human colorectal adenocarcinoma cells (DLD-1) and the kidney of an African green monkey cell derived (Vero) were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Gibco-Thermo Fisher Scientific, MA, USA) supplemented with 10% of inactivated fetal bovine serum (Gibco-Thermo Fisher Scientific, MA, USA). The cells were transferred to a 96-well microplate, so the initial cell number was 1.25 × 10⁴ cells 5 × 10³ cells for DLD-1 and Vero, respectively, per well, then incubated at 37 °C, 5% CO₂ for 24 h. The sample was added as much as 0.4 µL or a volume so the final concentration of DMSO in the culture was less than 1%, then incubated at 37 °C, 5% CO₂ for 48 h. After being washed with 100 µL of PBS, the cell was resuspended with 100 µL of DMEM containing 10% of Cell Counting Kit-8 (Dojindo, Kumamoto, Japan) and placed in the incubator at 37 °C, 5% CO₂ for 3 h. The absorbance of each well was measured at 450 nm by a plate reader (Spectramax Paradigm, Molecular Devices, San Jose, CA, USA).

DCs were generated as described previously by Oliveira et al. [29 (link)], with some modifications. Briefly, bone marrow obtained from femurs and tibiae removed from C57BL/6 mice were cultured in 10 ml RPMI-1640 (GE Healthcare, Uppsala, Sweden) supplemented with 10% v/v inactivated fetal bovine serum (Gibco, Grand Island, NY, USA), 50 mM 2-mercaptoethanol (Sigma), 1 mM sodium pyruvate (Sigma), 25 mM sodium bicarbonate (Gibco), 10 mM HEPES (Sigma), 100 UI/ml penicillin (Sigma), 100 μg/ml streptomycin (Sigma), 25 mM L-glutamine (Gibco), and murine GM-CSF (25 ng/ml). Cells suspensions were prepared at 2.0 × 10⁵ cells/ml. On the fourth day of culture, 10 ml of culture medium supplemented with GM-CSF (50 ng/ml) was added to the plate. After seven days of culture, cells were harvested and their phenotype determined according to expression of CD11b and CD11c by flow cytometry; experiments were continued only when a DC phenotype was confirmed.

Raw 264.7 cells—a mouse leukemic monocyte macrophage cell line (ATCC TIB-71)—were cultured (as described elsewhere [17 (link)]) in Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich, St. Louis, MO, USA), supplemented with: 10% inactivated fetal bovine serum (Gibco, Paisley, UK); 100 U/mL penicillin (Sigma-Aldrich, St. Louis, MO, USA); and 100 µg/mL streptomycin (Sigma-Aldrich, St. Louis, MO, USA), at 37 °C in a humidified atmosphere of 95% air and 5% CO₂. Throughout the experiments, cells were monitored by microscopy in order to detect any morphological change.

The human CRC cell lines SW620, LS174T, and LoVo were cultured in RPMI-1640 (Gibco, Life Technologies) supplemented with 10% inactivated fetal bovine serum (Gibco, Life Technologies). All cell lines were cultured in a humidified atmosphere with 5% CO₂ at 37°C. The pEX-2-C1 (Mock), pEX-2-β3GnT8, and pEX-2-β3GnT2 plasmids were constructed as previously described (Liu et al., 2014 (link)). The pSilenCircle-negative control (NC), pSilenCircle-β3GnT8 (si-β3GnT8), and pSilenCircle-β3GnT2 (si-β3GnT2) plasmids was established by GenePharma (Suzhou, China). Cells were collected 48 h for assays after transfection with Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, United States).