Fig. 2: BMP4 treatment affects chemosensitivity in an EGFR-dependent manner.
a Western blot analysis of biochemical markers of apoptosis. Immunoblots show the levels of cleaved caspase 3, caspase 7, and PARP (two exposures) in untreated, BMP4- and/or TMZ-treated glioma spheres. Detection of β-actin ensured equal protein loading. b Densitometric analysis of apoptotic markers (a). Quantifications of three (L0125 and L0615) or two (L0512 and L0627) independent experiments are shown. Data are presented as the means ± SD (log-scale). See the legend in Fig. 1f for more details. c Changes in the expression of cell cycle genes induced by BMP4 and/or TMZ treatments in L0125 and L0615 spheres. The heatmap (top) shows changes in the expression of the genes from the ‘cell cycle’ Gene Ontology category. Z-scores were computed separately for the two cell lines. An unsupervised clustering led to two main clusters, named cluster 1 and cluster 2, which are highlighted on the right side. The computed clusters showed associations with genes gathered as ‘regulation of cell cycle G1/S phase transition’ or ‘regulation of cell cycle G2/M phase transition’ Gene Ontology terms (bottom). P-values were computed using Fisher’s exact test. The red dotted line marks a P-value = 0.05. d Distribution of cells in cell cycle phases was determined by flow cytometry. The left panel presents the results for all cell cycle phases as the means ± 95% confidence intervals for the means computed from three independent experiments. P-values shown above the plots were computed using χ2 tests. The right panel shows the effects of selected treatments (BMP4 alone and BMP4 + TMZ) on the fraction of cells in G1 (top) or G2/M (bottom) phases. The effects are shown by the difference in means and 95% confidence intervals for the differences. Yellow and purple colors correspond to the L0125 and L0615 spheres, respectively. The statistical significance was tested with ANOVA and Duncan’s post hoc test. P < 0.05 is written in red. e Western blot analysis of cell cycle-related proteins in untreated and BMP4 ± TMZ-treated spheres. Levels of p21CIP1, p27KIP1, Cyclin B1, Cyclin D1, and phospho-Rb (Ser807/811) proteins were analyzed. β-actin was used as a loading control. f Densitometry analysis of immunoblots shown in e. Each bar and whisker represent the mean ± SD of two or three independent experiments (log-scale). The horizontal P-value corresponds to the comparison between BMP4- and BMP4 + TMZ-treated cells, which was computed with Duncan’s post hoc test.
