(A) HD:MB03 cells were treated with 10 nM triptolide, followed by RNA-seq and gene set enrichment analyses (n = 3). Heatmap displays triptolide-regulated gene expression hallmarks, with an arrow indicating MYC targets. (B) The Cavalli et al. 2017 data set was analyzed to correlate MYC targets hallmark expression with G3 MB patient survival using log-rank (Mantel-Cox) tests. (C) DepMap gene dependency analyses predicted G3 MB cell dependency of genes downregulated by triptolide in the L1000 triptolide transcriptional response signature in Figure 1A. Arrow highlights MYC. (D) G3 MB cells were treated with triptolide for 24 hours, and MYC expression was quantified by RT-qPCR (n = 3). Values were analyzed using 1-way ANOVA followed by Dunnett’s post hoc test. (E) Lysates of G3 MB cultures exposed to triptolide were immunoblotted for the indicated proteins. (F) mG3-2929 cells were electroporated with HA-MYC 72 hours prior to 50 nM triptolide treatment. Cell viability was assessed by MTT reduction 48 hours later, while MYC levels were measured by immunoblotting 72 hours after electroporation (n = 3). (G) mG3-2929 cells were transfected for 48 hours with MYC-targeting siRNA, scramble siRNA (siSC), or GFP control, and then treated with 50 nM triptolide. Cell viability was assessed by MTT reduction 48 hours later, and MYC levels were measured by immunoblotting 72 hours after transfection (n = 3). (H) SHH-MB47 cells received MYC vector via electroporation 72 hours prior to exposure to 50 nM triptolide. Cell viability was measured using CellTiter-Glo assay 48 hours later. MYC levels were assessed in similarly electroporated cells exposed to 100 nM triptolide for 16 hours (n = 3). Images of representative immunoblots are shown. Unless otherwise indicated, all results are presented as mean ± SEM of data normalized to DMSO, where statistical significance was assessed using 1-way ANOVA followed by Newman-Keuls post hoc test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.