Skip to main content
. Author manuscript; available in PMC: 2019 Sep 6.
Published in final edited form as: Nature. 2019 Mar 6;567(7748):341–346. doi: 10.1038/s41586-019-0993-x

Extended Data Figure 1. Gboxin, isolated from 200,000 compound screen, specifically inhibits GBM growth (HTS cells) but not MEFs or astrocytes.

Extended Data Figure 1.

a. Flow chart of the primary and secondary compound screens performed with HTS, MEF, astrocyte and neural/progenitor stem cells (NSCs). b. Representative live cell images show Gboxin specific toxicity of HTS cells. Cells were treated with DMSO, Gboxin (1 μM) or cycloheximide (CHX, 1 μM) for 3 days. n = 4. c. Cell viability assays for SVZ derived primary neural stem/progenitor cells (NSCs) and HTS cells treated with increasing doses of Gboxin indicate a therapeutic window for HTS cells. n = 3; mean ± SD. d. Cell viability assays show irreversible growth inhibition in HTS cells as early as 6-hours following 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 hours after initial compound treatment. n = 3; mean ± SD. e. Gboxin induces specific transcription alterations in HTS cells. mRNA specific RT-qPCR analyses in MEFs and HTS cells treated with DMSO or Gboxin for 12 hours. n = 2. f. mRNA specific RT-qPCR assays in HTS, MEF and astrocyte cells treated with DMSO or Gboxin (1 μM) for 12 hours demonstrate HTS specific up and down regulation of gene expression. Mean ± SD; n = 3. g. Representative western blot analyses with astrocyte cells treated with DMSO or Gboxin (1 μM) for 6 hours indicate no effect of Gboxin on expression of ATF4 and phospho-S6 (p-S6). n = 3. h. Representative western blot analyses using HTS cells exposed to DMSO or Gboxin (1 μM) detect ATF4 upregulation within 3 hours after Gboxin treatment. n = 2. i. HTS cell cycle progression analyzed by flow cytometry of cells treated with DMSO or Gboxin (1 μM) for 24 hours indicates an increase of G1/0:S ratio. 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 of mitochondrial membrane potential in HTS cells. Representative images for TMRE staining show HTS cell specific neutralization of mitochondrial membrane potential after an 18-hour incubation with Gboxin. n = 3. l. quantification for k. Mean ± SD.