Fb essence

293T-HEK cells were maintained in Dulbecco’s Modified Essential Medium (DMEM) with 10% FB Essence (VWR), 1% penicillin/streptomycin and L-glutamine.

U87MG cells were acquired from ATCC‐ and U87MG‐derived cell lines were maintained in DMEM/F12 with 10% FB Essence (VWR, Radnor, PA, USA). GBM4 was obtained from H. Wakimoto (Wakimoto et al., 2009).

Mouse brain microglia (BV2, EOC 20 CRL-2469), human microglial cells (HMC3, CRL-3304), human astrocytoma cells (U87MG, HTB-14), and African green monkey kidney cells (Vero cells, CCL-81) were obtained from American Type Culture Collection (ATCC). HMC3 cells were maintained in 1X Eagle’s Minimum Essential Medium (1X EMEM, Life Technologies, 670086) supplemented with heat-inactivated 10% fetal bovine serum (FBS, Gibco) and 1% penicillin/streptomycin (Corning, 30-002-CI). U87MG cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM, Quality Biological, 112-012-101) supplemented with 10% FBS, 1% penicillin/streptomycin, and 1% L-glutamine (Corning, 25-005-CI). Vero and BV2 cells were maintained DMEM supplemented with 5% FB Essence (VWR, 10799-390), 1% penicillin/streptomycin, and 1% L-glutamine. Cell cultures were grown at 37 °C and 5% CO₂.

Fetal bovine serum (FBS) was purchased from R&D Systems (cat# S11550) and FB Essence (FBE) was purchased from VWR (cat# 10803–034). Human plasma was provided by Dr. Michael Pichichero. Bovine serum albumin (BSA) was purchased from Fisher Scientific (cat #BP1600-100) and fatty acid-free BSA (FAF-BSA) was from Sigma (cat#126575). Proteinase K was from Takara Bio (cat#ST3041). Actinomycin D was from Fisher Scientific (#NC9856244). Lysophosphatidic acid (#L7260), Lysophosphatidylcholine (#L1881), Propidium Iodide (#P4170), and Centrifugal Filter Units (#UFC501008, #UFC505008, # UFC510008) were from Sigma. The MTT Cell Growth Assay Kit was from Fisher Scientific (#CT01).

The 4T1 cells were purchased from ATCC (Manassas, VA, USA) and cultured in RPMI 1640 (ThermoFisher, Waltham, MA, USA) supplemented with 1% pen/strep, 2 mM L-Glutamine, 1 mM Sodium Pyruvate, 10 mM HEPES, 0.05 mM β-mercaptoethanol, and 10% (by volume) FB Essence (VWR, Radnor, PA, USA). To grow syngeneic, orthotopic mammary tumors, female BALB/cJ mice (Jackson Laboratory, Bar Harbor, ME, USA) between four to six weeks of age were inoculated with 1.0 × 10⁴ 4T1 cells. Cells were washed and suspended in 100 µL 1X HBSS (Ca²⁺/Mg²⁺ free, ThermoFisher) before injection through the nipple of the upper-right mammary fat pad using an insulin syringe. Mice were monitored daily for availability of food and water and any signs or symptoms of peripheral infection or inflammation. Palpable tumors were routinely detected 2 weeks after injection, and tumor sizes were monitored by calipers. Final tumor volumes were calculated by volume = (3.14/6)width × length² [70 (link)]. In the 4T1 tumor time-course study, tissues were harvested at days 0, 7, 14, 21, and 28 post-injection (Figure 2A). On the day of tissue harvest, mice were euthanized by CO₂ asphyxiation and tumors weighed before further processing. All animal procedures were performed according to approved Institutional Animal Care and Use Committee protocols (numbers: 1906CE-RH-RM-22 and 1702C-NP-M-20).

HEK293T cells were purchased from ATCC (CRL-11268) and were maintained in high glucose DMEM (Thermo Fisher Scientific) supplemented with 10% FB Essence (VWR), 1× GlutaMAX^™-I (Thermo Fisher Scientific), 1× MEM Non-Essential Amino Acids (Thermo Fisher Scientific), and 100 U/ml penicillin and 100 μg/ml streptomycin mixture (Thermo Fisher Scientific). For plasmid and siRNA transfection, Lipofectamine 3000 and Lipofectamine RNAiMAX were used, respectively, according to the manufacturer’s protocols. TUT4 KO, TUT7 KO, and DIS3L2 KO cell lines were established by transfecting LentiCRISPR V2-sgRNA with puromycin resistant marker into HEK293T cells. DIS3L2-TUT4/7 TKO cell line was established by transfecting DIS3L2KO cells with TUT4 and TUT7 LentiCRISPRV2-sgRNAs with hygromycin and zeocin resistant marker, respectively. Single colonies were picked and screened after drug selection.