Fig. 4

PCI-24781 suppresses GBM cell proliferation and induces apoptosis. Cells were seeded in DMEM complete media. After 12 h, cells were treated with vehicle control, IC₂₅ of TMZ, IC₂₅ of PCI-24781, or TMZ and PCI-24781 for 48 h, and cultured up to 2 weeks in drug-free DMEM complete media. Colonies were fixed with methanol and stained with crystal violet, then dissolved in 10% acetic acid, and absorbance measured at 595 nm. Results represented as difference in percentage colony formation. A Representative images of colony formation assay. B Mean percent difference in colony formation in different drug treatment groups from three independent experiments. ANOVA was used to compare the colony formation variable on the natural log scale. Pairwise comparisons are adjusted with Tukey’s method. ‘*’ p ≤ 0.0001; ‘$’ p ≤ 0.01; ‘#’ p < 0.001 “*” significantly different compared to vehicle control; “$” significantly different compared to TMZ; “#” significantly different compared to PCI-24781. C-E PCI-24781 induces apoptosis in GBM cells. C Representative Annexin V and PI staining of vehicle control, TMZ, PCI, and TMZ + PCI treated U-118MG cells. D Quantification (percentage of) early and late apoptotic cells. Early and late apoptosis was compared between groups using ANOVA. Pairwise comparisons are adjusted with Tukey’s method. ‘*’ p < 0.03; ‘$’ p < 0.0001; ‘#’ p < 0.0001. “*” significantly different compared to vehicle control; “$” significantly different compared to TMZ; “#” significantly different compared to PCI-24781. E U87, U87EGFRvIII, U251, U251EGFRvIII, and U-118MG cells were incubated with the drugs mentioned above for 48 h, and lysates were subjected to immunoblot analysis to identify the levels of apoptotic proteins, cleaved PARP, and cleaved caspase-3