The GSC lines were established by isolating neurosphere-forming cells from fresh surgical specimens of human GBM tissue obtained from patients at The University of Texas MD Anderson Cancer Center from 2005 to 2008, as described previously.21 (link) Eight GSC lines [4 with MGMT-unmethylated/MGMT expression (MGMT+)] and [4 with MGMT-methylated/no MGMT expression (MGMT-)] were cultured in DMEM/F12 medium containing B27 supplement (Invitrogen), basic fibroblast growth factor, and epidermal growth factor. Short tandem repeats using the Applied Biosystems AmpFISTR Identifier kit were used to authenticate cells. The last authentication test was performed in July 2017. All cell lines tested negative for mycoplasma contamination using the MycoAlert Detection Kit.
TMZ was from Sigma–Aldrich and lomeguatrib, elimusertib, ceralasertib, and berzosertib were from Selleckchem. For in vitro use, all inhibitors were dissolved in dimethyl sulfoxide. For irradiation experiments, we performed photon irradiation using an X-RAD 320-Precision X-Ray Biological Radiator to deliver a precise dosage of 2 Gy to 12 Gy to the cultured cells. After irradiation, cells were transferred to a cell culture–grade incubator for downstream applications. Sham-treated cultures were kept in close proximity to the X-RAD device for the same amount of time without exposure to X-rays.
TMZ was from Sigma–Aldrich and lomeguatrib, elimusertib, ceralasertib, and berzosertib were from Selleckchem. For in vitro use, all inhibitors were dissolved in dimethyl sulfoxide. For irradiation experiments, we performed photon irradiation using an X-RAD 320-Precision X-Ray Biological Radiator to deliver a precise dosage of 2 Gy to 12 Gy to the cultured cells. After irradiation, cells were transferred to a cell culture–grade incubator for downstream applications. Sham-treated cultures were kept in close proximity to the X-RAD device for the same amount of time without exposure to X-rays.
