Human neurocult ns a proliferation supplements

The patient-derived GSCs were well characterized (Table 1) and cultured in NS-A medium (90% NeuroCult NS-A Basal Medium Human plus 10% Human NeuroCult NS-A proliferation Supplements, StemCell Technologies, Vancouver, British Columbia, Canada in 3D sphere as we described before [45 (link)]. Complete medium was supplied with recombinant human epidermal growth factor (R&D System, Minneapolis, MN, USA) and 100 units/mL penicillin-100 μg/mL streptomycin (Life Technologies, Carlsbad, CA, USA). Anti-TUSC3 and anti-MGMT antibodies were obtained from Abcam (Cambridge, United Kingdom); anti-β-actin IgG-HRP was obtained from Santa Cruz Biotech (Dallas, TX, USA) and anti-DNMT1, Goat anti-Rabbit IgG-HRP and Goat anti-mouse IgG-HRP were obtained from CellSignaling Technology (Danvers, MA, USA). Temozolomide (TMZ) was obtained from Sigma (St. Louis, MO, USA). 5-Azacitidine (5-Aza) and Lomeguatrib were obtained from Selleckchem (Houston, TX, USA).

U251 and U87 human glioblastoma cell lines (ATCC) were cultured in DMEM (Life Technologies) supplemented with 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA), 100 units/mL penicillin and -100 ug/mL streptomycin (Life Technologies, NY, USA). The cultures were maintained at 37 °C in a humidified atmosphere containing 5% CO₂. The patient-derived GSCs used in this study were isolated from GBM patients and were well characterized. The patient-derived GSCs were culture in NS-A medium (90% NeuroCult NS-A Basal Medium Human plus 10% Human NeuroCult NS-A proliferation Supplements, StemCell Technologies). Complete medium was supplied with recombinant human epidermal growth factor (R&D system, Minneapolis, MN, USA), and 100 units/mL penicillin plus 100 ug/mL streptomycin (Life Technologies, NY, USA). For differentiation, GSCs were cultured in NS-A medium supplied with 10% fetal bovine serum. Anti-BIRC3 antibody was obtained from R&D system; anti-b-actin IgG-HRP was obtained from Santa Cruz Biotech; and anti-SMAD1, anti-SMAD5, anti-p-SMAD1/5, Goat anti-Rabbit IgG-HRP and Goat anti-mouse IgG-HRP were obtained from CellSignal.