6 well cell culture cluster

Human MG-63 and U2OS osteosarcoma cell lines were purchased from Cells Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China). These cells were cultured in Dulbecco-modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Hyclone, Logan, UT), at 37 °C in a humidified atmosphere with 5% CO₂. MG-63 cells were plated onto 6-well cell culture clusters (Costar) and grown to 80% confluence, and then serum-starved for 24 h. These cells were subsequently treated with recombinant sfrp2 or Wnt5a (R&D Systems, Minneapolis, MN) or PI3K/Akt inhibitors (LY294002, HS-173, TGX-221, CZC24832, CAL-101, MK-2206, A-674563, CCT128930) (Selleck, Houston, TX) before small G-protein activation assays and cell migration assays.

In all, 1.0 × 10⁵ cells of glioma cell lines and GSCs were seeded in 6-well cell culture cluster (Corning, Corning, NY, USA), and cultured in 2.0 mL medium. The final concentration of treated [¹⁵N₅]-dA was 20 μM, and the final concentration of treated [D₃]-l-methionine was 30 μg/mL. The final concentration of treated [D₃]-m⁶A was ranged from 0.2 to 10 μM. The cells were treated with [¹⁵N₅]-dA or [D₃]-L-methionine for 4 days, or with [D₃]-m⁶A for 2 days and harvested for DNA extraction and UHPLC-MS/MS analysis.

Target tumor cell suspension was stained with 2.5 μM CellTracker Green CMFDA dye (Invitrogen, Carlsbad, CA) for 30 min at 37°C in the absence of serum. Monolayer of the tumor cells were incubated in DMEM with 10% FBS at a density of 3.5 × 10⁵ cells/well in 6 well cell culture cluster (Corning, Union City, CA) for 12 h at 37°C in order to adhere. Immune cells were stained with 10 μl CD45-PE (Beckman Coulter, Brea, CA) for 20 min at room temperature prior to co-incubations with adherent tumor cells by a density of 3.5 × 10⁵ cells/well at indicated time to allow CICs formation. Flow cytometry was gated by unstained/stained effector cells and tumor cells alone, and unstained effector/tumor cells co-cultures as well.

Human glioblastoma cell line U87MG (WHO grade IV) was purchased from the National Institute for Cancer Research of Genoa (Italy). The human T98G and U343MG cell lines (WHO grade IV) were obtained from the American Type Culture Collection (USA) and CellLines Service GmbH (Germany), respectively. Cell lines were controlled for DNA profiling. U343MG cells were maintained in Minimum Essential Medium Eagle with 2 mM l-glutamine (Sigma-Aldrich, Milan, Italy), 1.5 g/L sodium bicarbonate (Sigma-Aldrich, Milan, Italy), 10% FBS (Sigma-Aldrich, Milan, Italy), 100 U/mL penicillin, 100 mg/mL streptomycin (Sigma-Aldrich, Milan, Italy), 1% non-essential amino acids, and 1.0 mM sodium pyruvate (Sigma-Aldrich, Milan, Italy). U87MG and T98G cells were maintained in an RPMI medium supplemented with 10% FBS, 2 mM l-glutamine, 100 U/mL penicillin, 100 mg/mL streptomycin, and 1% non-essential amino acids (NEAA) (Sigma-Aldrich, Milan, Italy). HeLa cells were propagated in high glucose Dulbecco’s modified Eagle’s medium (DMEM) (Euroclone) with 10% foetal bovine serum (FBS) (Euroclone). Cell cultures were maintained on a 6-well cell culture cluster (Corning) at 35 °C with 5% CO₂. Mycoplasma contaminations were excluded by PCR using primers GPO-3 and MGSO.

After an interval of 5 days to allow for retrograde labeling surgery and 7 days for ONC surgery, the animals were deeply anesthetized as described above. The retina preparation process was similar to that previously reported [24 (link)]. Animals were euthanatized with an intravenous injection of pentobarbital sodium (>50 mg/kg) and perfused transcardially with 0.75 L of physiologic saline (0.9%), followed by 1.0 L of solution containing 4% paraformaldehyde (PFA) (#15710, Electron Microscopy Sciences, Ft. Washington, PA, USA) in 0.1 M phosphate buffered saline (PBS)(pH 7.4, Sigma-Aldrich, USA). The brains were placed in the same fixative for subsequent processing. The eyes were enucleated, and the lens and vitreous were carefully removed in 0.1 M cold PBS. The eyecups were fixed immediately by immersion in 4% PFA as described above for 60 min at room temperature. The retinas were then dissected from the retinal pigment epithelium, and 3–4 radial cuts were made to flatten the retina. The retina was then mounted on a nitrocellulose membrane filter (GSWP02500, Millipore, USA) and transferred to a 6-well cell culture cluster (#3516, Corning Incorporated, Corning, USA) for immunohistochemistry staining.