Ln229

Four IDH-wild type (WT) glioma cell lines (A172, U251, SHG44 and LN229) were bought from Procell (Wuhan, China) and human normal astrocyte cell line (NHA) was bought from Mingzhoubio (Ningbo, China). All cells were cultivated at 37 °C in Dulbecco’s modified Eagle’s medium (DMEM; Procell) mixed with 10% fetal bovine serum (FBS; Procell) and 1% penicillin–streptomycin solution (Procell) in a humid incubator including 5% CO₂.
To block transcription, A172 and U251 cells were exposed to Actinomycin D (2 μg/mL; Sigma-Aldrich, St. Louis, MO, USA) for 8 h, 16 h and 24 h. Afterward, the levels of circRFX3 and RFX3 mRNA were examined using the quantitative real-time polymerase chain reaction (qRT-PCR) analysis mentioned next.

The glioma cell lines (U118, U138, LN229, and HS683) and their culture medium (Dulbecco’s modified Eagle medium) were obtained from Procell Life Science & Technology Co., Ltd. (Wuhan, China). HA and its culture medium (Astrocyte Medium) were purchased from ScienCell Research Laboratories, Inc. (San Diego, CA, USA). All the cells were cultured in a sterile cell incubator at 37 °C with 5% CO₂.

Normal human astrocytes (NHAs), U251, A172, T98G, LN229, U118 and THP‐1 cells lines were purchased from Procell. NHAs and glioma cell lines were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Sciencell). The THP‐1 cells were cultured in RPMI 1640 medium with 10% heat‐inactivity FBS. Besides, 0.1 μg/ml phorbol‐12‐myristate‐13‐acetate (PMA, Sigma Aldrich) was used to induce differentiation of THP‐1 cells into macrophages. All cells were placed in an incubator with an atmosphere of 5% CO₂ at 37°C.

Normal human astrocytes and glioma cell lines LN229, T98, U251, and A172 were purchased from Procell Life Science & Technology Co, Ltd. (Hyderabad, India). All cells were incubated in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, USA) at 37°C under 5% CO₂ in a humidified incubator. Expression of CKAP2L was measured in all five cell lines, as described below, and the cell line with the highest CKAP2L expression was selected for further experiments. Accordingly, U251 cells were transfected with lentiviruses containing control shRNA or shRNA against CKAP2L (5′-GATCCGCAAACAAAGAGAACTTGCTCGATATTTCAAGAGAATATCGAGCAAGTTCTCTTTGTTTGTTTTTTGGAAG-3′), which were purchased from GenePharma (Suzhou, China), and screened using puromycin.

Human glioma cell lines U87, T98G, LN229, U373, human astrocyte cell line HA1800, and human mononuclear macrophage line (THP-1) were purchased from Procell Life Science & Technology (Wuhan, China). U251 was purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Murine glioma cell line GL261 was obtained from American Type Culture Collection (Manassas, VA, USA). All cell lines were tested without mycoplasma contamination. The human and murine glioma cells were cultured in Dulbecco’s Modified Medium (DMEM) with 10% fetal bovine serum (FBS). THP-1 was maintained in RPMI-1640 medium with 10% FBS. All the cell culture media were supplemented with 1% penicillin/streptomycin (10378016, Gibco) to prevent bacterial contamination.THP-1 monocytes were conditioned with 5 nM PMA (P1585, Sigma) for 48 h to differentiate as THP1-derived macrophages. All cells were cultured at 37 °C in a humidified incubator with 5% CO₂.