U87mg

The human GBM cell lines U87MG and U118MG were purchased from the American Type Culture Collection (ATCC). U87MG and U118MG was maintained in Minimum Essential Medium (MEM) and Dulbecco’s modified Eagle’s medium (DMEM), respectively, supplemented with 10% fetal bovine serum (Gibco, United States), 100 μg/ml streptomycin, and 100 U/ml penicillin, at 37°C in 5% CO₂. Three small interfering RNAs (siRNAs) against METTL3 were used to inhibit METTL3 expression. siCrtl was used as a control. METTL3 siRNA sequences were shown in Supplementary Table S1. All siRNAs were designed and chemically synthesized by Obio Technology (Obio Technology Co. Ltd., China). siRNAs were transfected into U87MG and U118MG using X-treme GENE^TM HP DNA Transfection Reagent (Roche, Switzerland) according to the manufacturer’s instructions. Cells were harvested for RNA and protein extraction assay at 48 h after the transfection.

The cell lines were obtained from ATTC (Manassas, VA, USA). HeLa (human cervix epithelioid carcinoma), MCF7 (human breast adenocarcinoma), U87 MG (human glioblastoma), T98G (TMZ-resistant human glioblastoma), HepG2 (human hepatocellular carcinoma), and HEK-293 (human embryonic kidney) were grown in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Waltham, MA, USA) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (HIFBS, Sigma-Aldrich, St. Louis, MO, USA), 2 mM L-glutamine, 100 μg/mL streptomycin, and 100 units/mL penicillin (Gibco, Waltham, MA, USA) at 37 °C, 95% humidity, and 5% CO₂. The HT-29 and HCT8 (human colon carcinoma) cell lines were grown in Roswell Park Memorial Institute 1640 (RPMI 1640) (Gibco, Whaltman, MA, USA) supplemented with 10% HIFBS, 100 μg/mL streptomycin, and 100 units/mL penicillin (Gibco, Whaltman, MA, USA) at 37 °C, 95% humidity, and 5% CO₂. The cells were periodically tested for Mycoplasma infection by using a MycoAlert kit (Lonza, Norwest, Australia), and only Mycoplasma-free cells were used in the experiments.

GBM 8401 (BCRC, CVCL_B051), U87 MG (ATCC^® HTB14), T98G (ATCC^® CRL-1690), and U138 MG (ATCC^® HTB-16) were the commercial GBM cell lines of Bioresource Collection and Research Center (Hsinchu, Taiwan) or American Type Culture Collection (Manassas, VA, USA). Alpha-MEM medium (Gibco BRL, Rockville, MD, USA) was used for the maintenance of U87 MG, T98G, and U138 MG, while GBM 8401 cells were cultured in RPMI-1640 medium (Gibco). These cells were supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin (Gibco) and cultured under a humidified atmosphere of 5% CO₂ and 95% room air at 37 °C. EA.hy926 cells (ATCC^® CRL2922™) was an established human fusion cell line by fusing primary human umbilical vein cells with a thioguanine-resistant clone A549. The fusion cells were cultured in Dulbecco’s Modified Eagle’s Medium with 10% of fetal bovine serum and 100 U/mL of penicillin and streptomycin and cultured under the same atmosphere as for the GBM cells.

The human prostate cancer cell line PC-3, pharynx carcinoma cell line FaDu, and human glioblastoma astrocytoma cell line U87-MG were purchased from the American Type Culture Collection (ATCC). The colorectal adenocarcinoma cell line HT-29 was obtained from the European Collection of Authenticated Cell Cultures (ECACC). The amelanotic melanoma cell line MV3 and the breast cancer cell line MDA-MB-231 were purchased from EPO GmbH Berlin-Buch. The human AsPC1 cell line was provided by Prof. Dr. Ulrich Massing, University Hospital Freiburg, Germany, Clinic for Tumor Biology. Cell culture materials and reagents were purchased from PAN Biotech (Aidenbach, Germany) unless otherwise stated. All cells were incubated in a humidified atmosphere with 5% CO₂ at 37 °C in RPMI 1640 (PC-3, MV3, AsPC1, FaDu), DMEM (MDA-MB-231, U87-MG) or McCoy´s 5A medium (HT-29) containing 10% fetal bovine serum and 2 mM L-glutamine. Medium was supplemented with 50 U/mL penicillin and 50 µg/mL streptomycin (PC-3, MV3, AsPC1, MDA-MB-231) and 1 mM sodium pyruvate (PC-3, MDA-MB-231). FaDu, HT-29, and the U87-MG cell line medium was purchased from Gibco (Darmstadt, Germany); the corresponding fetal bovine serum was purchased from Sigma Aldrich (St. Louis, MO, USA).

Human glial tumour cell lines A172 and U‐87 MG were obtained from The European Collection of Authenticated Cell Cultures (ECACC, Sigma‐Aldrich). Canine glial tumour cell lines J3T‐BG, SDT3G and G06A were a kind gift of PD Philippe Plattet, PhD (Division of Neurological Sciences, Vetsuisse Faculty, University of Bern, Bern, Switzerland) and were previously described (York et al., 2012 (link)). A172 cells were maintained in DMEM cell culture medium (Gibco) supplemented with 10% FBS (Gibco), 100 units/ml of penicillin (Gibco), 100 μg/ml of streptomycin (Gibco) and incubated at 37°C in 5% CO₂ humidified incubator. U‐87 MG cells were maintained in EMEM cell culture medium supplemented with 1% Non‐Essential Amino Acids (NEAA) (Gibco), 10 mM HEPES (Gibco), 10% FBS (Gibco), 100 units/ml of penicillin (Gibco), 100 μg/ml of streptomycin (Gibco) and incubated at 37°C in 5% CO2 humidified incubator. J3T‐BG, SDT3G and G06A cells were grown in RPMI 1640 (Gibco) supplemented with 10% FBS (Gibco), 100 units/ml of penicillin (Gibco), 100 μg/ml of streptomycin (Gibco) and incubated at 37°C in 5% CO₂ humidified incubator.

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.