Generation and Validation of GBM Cell Lines

SJ-GBM2 cells (CVCL_M141) were a gift of the COG Repository at the Health Science Center, Texas Tech University (Lubbock, Texas, USA). SJ-GBM2 cells were grown in IMDM (Gibco, Thermo Fisher Scientific, catalog 1244005320) supplemented with 20% FBS at 37.0°C, 5% CO₂, 20% O₂ according to a previously published report (27 (link)) and were used in early passages and tested regularly for mycoplasma. SJ-GBM2 cells were transfected using jetPRIME (Polyplus, catalog 114-01) with the plasmids pLVX-BFP-H3.3WT and pLVX-BFP-H3.3G34R described above (Supplemental Figure 4). After transfection, cells were allowed to grow for 4 days, and selected with puromycin (10 μg/μL, Goldbio, catalog P-600-10). After amplification in the presence of selection, cells were subjected to FACS for isolation of BFP⁺ cells. Three independent polyclonal populations were obtained for each genotype (SJ-GBM-2-H3.3-G34R and SJ-GBM-2-H3.3-WT). These cells are referred to as H3.3-G34R and human pHGG cells.