Fig. 4
ISP I triggers ROS accumulation and oxidative stress response pathways. A Glutathione peroxidase activity assay shows a dose-dependent decrease of anti-oxidative enzyme activity in LN229 cells treated with ISP I. B Quantification of intracellular reactive oxygen species (ROS) levels by ROS-Glo H2O2 assay. LN229 and U251 cells were treated with ISP I at 10 μM for 24 h. All groups were normalized to saline-treated groups. C Flow cytometry analysis of ROS level using MitoSOX staining in ISP I-treated LN229 and U251 cells. Cells were pre-treated with 5 μM or 10 μM ISP I for 24 h. D Summarized ROS levels by mean fluorescence intensity (MFI) of MitoSOX positive cells from 3 independent wells are shown. E and F Transcriptomic profiling reveals that ISP I provokes an oxidative stress response pathway. E Gene Set Enrichment Analysis (GSEA) showing that ISP I-treated cells are highly enriched in genes associated with oxidative stress response. NES = 2.02. P < 0.05. F Heatmap of statistically significant differential gene expression as determined by RNA-seq between saline-treated and ISP I-treated cells. LN229 Cells were treated with ISP I at 10 μM or saline for 6 h. N = 4. P < 0.05. G Real-time RT-PCR analysis shows NFE2L2 mRNA expression in glioblastoma cell lines (LN229 and U251) treated with 10 μM ISP I for 24 h. 18S expression serves as an internal control. H Real-time RT-PCR results show the mRNA expression of NFE2L2 downstream gene-HMOX1 and NQO1 with 18S as an internal control. I Western bots show NFE2L2 and SELH protein expression in LN229 and U251 cells with SELH overexpression or with knocking down of SELH. GAPDH expression was used as an internal control. J and K Real-time RT-PCR results show NFE2L2 mRNA (J) and its downstream gene HMOX1 mRNA (K) in SELH knockdown LN229 and U251 cells. All data are shown as mean ± SEM. P value: *p < 0.05; **p < 0.01; ***p < 0.001