Control lentivirus

Overexpressing lentiviruses for OGDHL (NM_018245), FASN (NM_004104), and control lentiviruses were purchased from GeneChem Corporation (Shanghai, China). The vector backbone for overexpressing lentivirus is “Ubi-MCS-3FLAG-SV40-EGFP-IRES-Puromycin”. A498 cells and CAKI cells were infected to establish stable lines according to the manufacturer’s instructions. Puromycin (2 μg/mL, Sigma, USA) was used in established stable cell lines for further cell selection.
The OGDHL siRNA, FASN siRNA, and FTO siRNA were purchased from Genepharma Corporation (Shanghai, China). FTO and TFAP2A overexpression plasmids were purchased from GeneChem Corporation (Shanghai, China). Cell lines were transfected according to the manufacturer’s instructions, and cell experiments were completed within 24–120 h.

pLenti-U6-anti-miR-6359-CGGGAGC-EF1N promoter-Lamp-2b (CX3CL1-Extra- sv40-puro)-sv40-puro lentiviruses and control lentiviruses were bought from GeneChem (Shanghai, China). They were used to transfect SVEC (mouse lymphatic endothelial cells, ECs). Briefly, the lentiviruses were seeded in SVEC with a multiplicity of infection (MOI) of 20, and the corresponding empty vectors were used as controls. Cells overexpressing CX3CL1 and anti-miR-6359-CGGGAGC were harvested following 4~6 days of puromycin (Sigma-Aldrich) selection at a final concentration of 2 μg/ml.

Normal neurogliocyte (NHA) and four GBM cell lines (U87, T98, A172 and LN229) were obtained from the American Type Culture Collection. All cells were maintained in RPMI-1640 medium (Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco). All cells were cultured in a controlled environment with 5% CO₂ at 37 °C. The authenticity of all cell lines was confirmed through STR profiling. The FOXP3-overexpression lentivirus, short hairpin RNAs targeting FOXs, and their corresponding control lentiviruses were obtained from Genechem (Shanghai, China). The transfection of lentivirus was conducted using polybrene. A total of 0.5 µg/mL puromycin was used to select cells with stable FOXP3-overexpression or FOXP3-knockdown after 48 h transfection. We obtained small interfering RNA (siRNA) mimics targeting GPX4 and Linc00857 from iGenebio (Beijing, China). Transfecting mimics and siRNAs was performed using Lipo2000 (Thermo Fisher Scientific, USA) according to the manufacturer’s instructions. Sequences of shRNAs, mimics and siRNAs were showed in Supplement Table 1.

For gene knockdown, sh-LRP1 and control lentiviruses were synthesized by Genechem Co., Ltd. (Shanghai, China). Cells were seeded into six-well plates for 1 day. The culture medium was then refreshed; cells were transfected with sh-LRP1 or control lentiviruses for 3 days. Gene-silencing efficiency was validated by Western blotting. The shRNA sequences are shown in Supplementary Table S1.