U251mg

The human astrocytoma cell line U251 MG (KO) was purchased from the JCRB Cell Bank (Tokyo, Japan). U251 MG (KO) cells were cultured in E-MEM containing L-glutamine, phenol red, sodium pyruvate, nonessential amino acids, and 1,500 mg/l sodium bicarbonate (Wako, Osaka, Japan; #055–08975) supplemented with 10% fetal bovine serum (Life Technologies; #10437085) and 100 U/ml penicillin–streptomycin (Life Technologies; #15140122) under 5% CO² at 37°C. To obtain cells stably overexpressing Venus-tagged FUS and mCherry-tagged G3BP1, the appropriate expression vectors were introduced into cells with Lipofectamine 3000 (Life Technologies) in Opti-MEM I (Thermo Fisher Scientific, Waltham, MA; #31985070) following the manufacturer’s instructions. U251 MG (KO) cells positive for Venus and mCherry signals were selected with 100 μg/ml G418 (geneticin; Thermo Fisher Scientific). To induce DNA damage, cells were treated with 10–100 nM CLM (MedChem Express, Monmouth Junction, NJ; #HY19609) with or without 1–10 μM NU7441 (Wako; #143-09001), a high-potency selective DNA-PK inhibitor.

Glioblastoma cell lines IN-GB-11, IN-GB-28, IN-GB-29, and IN-GB-9 were established from fresh tumor biopsies according to standard cell culture techniques [18 (link)] at the Institute of Neurology, Medical University of Vienna. All typical mutations (e.g., TP53, PTEN) present in the cell lines were confirmed by mutation analysis of the corresponding tumor tissue. Human primary astrocytes (HPAs) were purchased from Applied Biological Materials (ABM) Inc. (ABM Inc., Richmond, BC, Canada). HPAs were cultured on collagen-coated dishes in Prigrow IV medium (ABM Inc., Richmond, BC, Canada), supplemented with 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA). Human glioblastoma cell lines U87-MG, U251-MG, and human embryonic kidney 293T cells (HEK293T) were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). U251-MG and HEK293T cells were cultured in Dulbecco’s modified Eagle medium (DMEM; Gibco, Grand Island, NY, USA), and U87-MG cells were cultured in Minimum Essential Media (MEM; Gibco, Grand Island, NY, USA). Both media, DMEM and MEM, were supplemented with 10% FBS (FBS; Gibco, Grand Island, NY, USA), 100 mg/mL penicillin G, and 100 lg/mL streptomycin (Gibco, Grand Island, NY, USA). All cell lines were cultured at 37 °C in a humidified environment with 5% CO₂.

The glioblastoma cell lines T98G, DBTRG-05MG, U251MG, and U87MG were purchased from the European Collection of Cell Cultures (ECACC). T98G cells were grown according to the manufacturer’s protocol in Minimum Essential Media (MEM; Thermo Fisher Scientific, Naarden, The Netherlands) supplemented with 1% non-essential amino acids (NEAA; Gibco, Ghent, Belgium), DBTRG-05MG in RPMI 1640 supplemented with 1 mM sodium pyruvate (Gibco, Ghent, Belgium), whereas U251MG and U87MG in Eagle’s Minimum Essential Media (EMEM) supplemented with 1% NEAA and 1 mM sodium pyruvate. For all cell lines, the media were also complemented with 1% L-glutamine (Thermo Fisher Scientific, Naarden, The Netherlands), 10% heat-inactivated fetal bovine serum (FBS; Thermo Fisher Scientific, Naarden, The Netherlands), and 1% Antibiotic-Antimycotic (100 units/mL of penicillin, 100 µg/mL of streptomycin and 0.25 µg/mL of Fungizone; Thermo Fisher Scientific, Naarden, The Netherlands). Cells were incubated at 37 °C in a humidified atmosphere of 5% CO₂.

The glioblastoma cell lines U251MG and U87MG were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). U251MG and U87MG cells were cultured in complete minimum essential media (MEM) (GibcoTM, Bleiswijk, NL) supplemented with 10% FBS (Gibco, Bleiswijk, NL) and 1% Pen/Strep (Gibco, Bleiswijk, NL). Maintained in a humidifying incubator at 37 °C with 5% CO₂, the cells were regularly tested for mycoplasma contamination.

U87MG, U251MG, U373MG, MCF7, PC3, B16F10, and B16F1 cells were purchased from the American Type Culture Collection (VA, USA). All cells were maintained at 37 °C in a humidified 5% CO₂ environment. Cells were cultured in minimal essential medium (MCF7), RPMI (PC3, B16F10, B16F1), or Dulbecco's modified Eagle's medium (U87MG, U251MG, U373MG) supplemented with 10% fetal bovine serum (FBS; Gibco, IL, USA), 100 U/mL penicillin (Gibco), and 100 µg/mL streptomycin (Gibco). All cell culture media were purchased from Gibco.

The two rat glioma C6 cell lines (named as C6-1 and C6-2 in our laboratory) were originally obtained from the National Health Research Institute Cell Bank (Zhunan, Taiwan) or the American Type Culture Collection (ATCC; Manassas, VA, USA), respectively. The high tumorigenic C6-1 cells with enriched IL-33 expression and low tumorigenic C6-2 with extremely low IL-33 expression have been previously characterized in vitro and in vivo (Fang et al., 2014 (link)). These cells were grown in culture flasks in Dulbecco's Modified Eagle's medium (DMEM)/F12 (Gibco, Grand Island, NY, USA), containing 10% heat-inactivated fetal bovine serum (FBS; HyClone, Logan, UT, USA). Alternatively, human malignant glioma cell lines, U251MG and U87MG, originally from ATCC were maintained in DMEM medium (Gibco) containing 10% FBS. The culture medium and antibiotics were purchased from Invitrogen (Carlsbad, CA, USA).

Two human glioblastoma cell lines (U118 MG, U251 MG) were used as an in vitro model. The U-118 MG was obtained from the ATCC^® (American Type Culture Collection, Virginia, No. HTB-15™) and was continuously cultured in Dulbecco’s modified Eagle’s medium (DMEM, ATCC^®, No. 30-2002). The U251 MG (No. 09063001) human glioblastoma astrocytoma cell line was purchased from PHE (Public Health England, Salisbury, UK) culture collections and cultured in Dulbecco’s modified Eagle’s medium (DMEM, high glucose, GlutaMAX™, Gibco^®, No. 31966021, Thermo Fisher Scientific, Waltham, MA, USA). The cells were maintained in 25 cm² tissue culture flasks (TPP^®, No. 90026, ) and the cultures were supplemented with 10% fetal bovine serum (FBS, Gibco^®, No. 10270106), in a humidified incubator at 37 °C with 5% CO₂. When 70% confluent cell monolayer was reached, the cells were detached with Trypsin/EDTA solution 1× (Sigma-Aldrich. No. T4049, Darmstadt, Germany) until complete cell detachment. The cell suspension with trypsin inactivated by a 5-fold dilution with fresh growth medium was spun down (3 min, 700 rpm) in 15 mL conical tubes, and the cell pellet was then resuspended in a fresh growth medium for cells scoring.