Fig. 4: Involvement of the AKT/FOXO3a/BIM pathway in TMZ-induced apoptosis of BMP4-differentiated EGFR^high GSCs.

a A heatmap representing RNA-seq expression of AKT1, FOXO3, and BCL2L11 genes in EGFR^low (L0125, yellow) and EGFR^high (L0615, purple) untreated glioma spheres. The results are presented on log scale. P-values shown above the heatmap were computed using the DESeq method and adjusted with the Benjamini and Hochberg method. The result of unsupervised clustering is shown on the right. b A densitometric analysis of immunoblots (c) shows the total and phospho-AKT (Thr308 and Ser473), total and phospho-FOXO3a (Thr32, Ser253, and Ser318) and BIM levels in untreated L0125 and L0615 spheres. Left, middle, and right panels correspond to total, phospho-AKT, and phospho-FOXO3a protein levels, respectively. Each bar and whisker represent the mean ± SD of three independent experiments. The total protein levels were normalized to those of β-actin, while the levels of phosphorylated proteins were normalized to the total level of the corresponding protein. Statistical analysis is shown in Fig. 1. c Western blot analysis of EGFR, total- and phospho-AKT, total- and phospho-FOXO3a and BIM levels upon exposure to TMZ, BMP4, or both drugs. β-actin was used as a loading control. d Densitometric analysis of phospho-AKT (Thr308) and phospho-FOXO3a (Thr32). Each bar and whisker represent the mean ± SD of three independent experiments (log-scale). The levels of phosphorylated proteins were normalized to the corresponding total protein levels. See legend for Fig. 1f for more details. The horizontal P-values correspond to comparisons between TMZ- and BMP4 + TMZ-treated cells and were computed with the post hoc test. e BCL2L11 gene expression in EGFR^low and EGFR^high cells after the treatments. The left and right panels correspond to L0125 and L0615 glioma spheres, respectively. The expression estimations are average values from the RNA-seq data of two independent experiments. The dashed lines correspond to TMZ treatment alone, while the solid lines correspond to pretreatment with BMP4 and subsequent treatment with TMZ. The adjusted P-value shown above the panels was computed using DESeq and was adjusted using the Benjamini and Hochberg method. f Densitometric analysis of BIM protein levels (n = 3). The bars and whiskers represent the means and SD (log-scale). The total protein levels were normalized to those of β-actin. See legend for Figs. 1f and 4d for more details. g A schematic summary of the obtained results. The left and right panels correspond to EGFR^low and EGFR^high glioma spheres, respectively. In EGFR^low cells, BMP4 stimulation triggers G1 cell cycle arrest, which leads to low BCL2L11 expression and blocks TMZ-triggered cell death. In contrast, TMZ treatment of BMP4-differentiated EGFR^high cells upregulates the level of the pro-apoptotic protein BIM via AKT inactivation, increases FOXO3a dephosphorylation and promotes its translocation into the nucleus.