Extended Data Fig. 1: Gboxin, isolated from a 200,000-compound screen, specifically inhibits GBM growth (HTS cells) but not that of MEFs or astrocytes.
From: Gboxin is an oxidative phosphorylation inhibitor that targets glioblastoma
a, Flow chart of the primary and secondary compound screens performed with HTS, MEF, astrocyte and neural stem and progenitor cells (NSCs). b, Representative live cell images show Gboxin-specific toxicity to HTS cells. Cells were treated with DMSO, Gboxin (1 μM) or cycloheximide (CHX, 1 μM) for three days. n = 4. c, Cell viability assays for subventricular-zone-derived primary neural stem and progenitor cells and HTS cells treated with increasing doses of Gboxin indicate a therapeutic window for HTS cells. Mean ± s.d., n = 3. d, Cell viability assays show irreversible growth inhibition in HTS cells as early as 6 h after Gboxin (1 μM) exposure. Cells were exposed to Gboxin for the indicated time periods, followed by culturing in Gboxin-free medium. Assay was performed 96 h after initial compound treatment. Mean ± s.d. n = 3. e, Gboxin induces specific transcription alterations in HTS cells. mRNA-specific quantitative PCR with reverse transcription (RT–qPCR) analyses in MEFs and HTS cells treated with DMSO or Gboxin for 12 h. n = 2. f, mRNA-specific RT–qPCR assays in HTS cells, MEFs and astrocytes treated with DMSO or Gboxin (1 μM) for 12 h demonstrate HTS-specific upregulation and downregulation of gene expression. Mean ± s.d., n = 3. g, Representative western blot analyses with astrocytes treated with DMSO or Gboxin (1 μM) for 6 h indicate no effect of Gboxin on expression of ATF4 and phospho-S6. n = 3. h, Representative western blot analyses using HTS cells exposed to DMSO or Gboxin (1 μM) detect ATF4 upregulation within 3 h of Gboxin treatment. n = 2. i, HTS cell cycle progression analysed by flow cytometry of cells treated with DMSO or Gboxin (1 μM) for 24 h indicates an increase in the ratio of G1 and G0 to S phase cells. j, Representative western blot analyses for proteins involved in apoptosis and survival with HTS cells treated with DMSO or Gboxin (1 μM) for 3 days. n = 3. k, Extended Gboxin exposure causes reduction in mitochondrial membrane potential in HTS cells. Representative images for TMRE staining show HTS-cell-specific neutralization of mitochondrial membrane potential after an 18-h incubation with Gboxin. n = 3. l, Quantification for k. Mean ± s.d.
