Rpmi 1460 medium

The human colon cancer cell line GEO was kindly provided by Dr. N. Normanno (National Cancer Institute, Naples, Italy). The human LIM1215 and SW48 colon cancer cell lines were purchased from the American Type Culture Collection (ATCC). The GEO-CR, SW48-CR and LIM1215-CR cell lines were established as previously described [15 (link),16 (link),29 (link),55 (link)]. The LIM1215, LIM1215-CR, SW48 and SW48-CR cancer cells were grown in RPMI-1460 medium (Sigma-Aldrich, Milan, Italy), whereas GEO and GEO CR cells were grown in McCoy’s 5A Modified Medium (Sigma-Aldrich, Milan, Italy) supplemented with 10% fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific, Rodano MI, Italy), 1% penicillin/streptomycin (Sigma-Aldrich). All cell lines were maintained at 37 °C in a humidified atmosphere with 5% carbon dioxide (CO₂). All cell lines were routinely screened for the presence of Mycoplasma using PlasmoTest-detection kit (InvivoGen, Toulouse, France).

PBMC were resuspended in complete medium (CM) in 10⁶/mL-concentration. This CM consisted of RPMI 1460 medium (Sigma-Aldrich, Taufkirchen, Germany) supplemented with 1% penicillin- streptomycin-L-glutamine (Sigma-Aldrich). Isolated PBMC were incubated at 37°C, with 1.5% phytohemagglutinin (PHA, 15 μg/mL) (Gibco, Gaithersburg, MD, USA). After 48 hours of incubation with PHA and exposure to humidified (95% H2O + 5% CO2) atmosphere, the supernatant was collected and stored at -70°C until enzyme-linked immunosorbent assays (ELISA).

ACHN and 786-O human kidney cancer cell lines were purchased from American Type Culture Collection (Manassas, VA). Cells were maintained 37°C in the presence of 5% CO₂ in Dulbecco’s modified Eagle’s medium/Ham’s F-12 or RPMI-1460 medium (Sigma-Aldrich, St. Louis, MO) with 10% fetal bovine serum. Sources of the antibodies used are summarized in Table 1. Cells were visualized under an EVOS fl, Fluorescence microscope, from Advanced Microscopy Group using a multiband filter set for FITC, rhodamine, and DAPI. The C-DIM compounds were prepared as previously described [9 (link)–11 (link)]. ACHN and 786-O kidney cancer cells (1.0 x 10⁵ per well) were plated in 12-well plates and allowed to attach for 24 hr, and treatment or knockdown experiments and immunofluorescence of NR4A1 was determined essentially as described [15 (link)–17 (link)].