Transcriptome U87MG TMZ resistant

Differential transcriptome analysis between control cells (U87MG), TMZ-resistant cells with continuous TMZ treatment (U87MG R50) and TMZ-resistant cells with interrupted treatment (U87MG OFF R50). Overall design: The U87MG GBM cell line was obtained from the American Type Culture Collection (LGC Standards Sarl, Molsheim, France). Two TMZ-resistant clonal lines were established in our laboratory by exposing the U87MG parent line to 50 µM TMZ for an extended period: U87MG R50 and U87MG R50 OFF. U87MG R50 cells were continuously cultured in a medium supplemented with 50 µM TMZ while U87MG R50 OFF were relieved of TMZ pressure after a two-month treatment period. TMZ (cat #T2577; Sigma Aldrich) was prepared as a 100mM stock solution in DMSO and stored at 4°C until use. These cell lines were routinely cultured in Eagle's Minimum Essential Medium (EMEM) with 10% heat-inactivated foetal bovine serum (FBS), 1% sodium pyruvate, and 1% non-essential amino acids at 37°C with 5% CO2. Glioma stem-like cells, NCH644 and NCH421k, were provided by Dr. Christel Herold-Mende (ref). Cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with Nutrient Mixture F-12 and GlutaMAXTM (DMEMF12+GlutaMAXTM, GibcoTM). These cells were cultured under serum-free conditions as neurospheres (NS) to maintain a stem-like state using the medium further enhanced with Bovine Serum Albumin (BSA) Insulin Transferrin (BIT-100, 20%) (Provitro), along with Epidermal Growth Factor (EGF, 20 ng/mL) and Fibroblast Growth Factor (ß-FGF, 20 ng/mL) (Reliatech). Induction of differentiation was achieved by culturing the cells in DMEM/F12 + GlutaMAXTM medium (GibcoTM) supplemented with 10% (vol/vol) FBS (GibcoTM) or all-trans retinoic acid (ATRA). The cultures were maintained at 37°C within a humidified atmosphere comprising of 5% CO2. Periodic authentication of all cell lines was conducted by Multiplexion GmbH, coupled with regular testing for mycoplasma contamination to ensure culture integrity