Figure 5

Validation of pathway interactomes predicted for ‘survival’ and ‘apoptosis’ processes. (A) GDC-0941-treatment inhibits downstream kinase AKT. Phosphorylation of AKT (S473) is inhibited after 4 h upon 5 µM GDC-0941-treament in A1 and WA01 cells. (B) Functional effect of GDC-0941-titrations on survival. Percentage of cells expressing the proliferation marker Ki67 decreases in a dose-responsive manner in WA01 and iPS-IMR90-1 cells after 24 h. (C) Functional effect of GDC-0941-titrations on apoptosis. Percentage of cells expressing active caspase-3+ is increased in WA01 and iPS-IMR90-1 cells in dose-responsive manner. (D) Validation of PI3Kα-knockdown efficiency in A1, WA01 and iPS-IMR90-1 cells. (E) Functional effect of PI3Kα-knockdown on apoptosis. Proportion of active caspase-3+ cells increased 2-fold upon PI3Kα-knockdown in WA01 and iPS-IMR90-1 cells. Bars represent means ± SEM of three independent experiments. (F) Representative FACS-plots showing percentage of active caspase-3+ WA01 cells upon PI3Kα-knockdown. (G) Cell cycle plots showing an increase in G1-phase in WA01, but not in A1 cells, after treatment with Entinostat (upper panels). FACS-histograms of CyclinD1 expression in WA01 (left) and A1 cells (right) before and after treatment with Entinostat (lower panels). The percentage of cyclinD1-expressing cells is increased in WA01 cells. (H) Fold increase in the percentage of G1-cells in WA01 and iPS-IMR90-1 cells upon 12 h Entinostat-treatment. (I,J) Notch1 and HDAC1-expression levels (MFI) in G1-cells correlate inversely with increasing Entinostat concentrations in WA01 cells (I), but not in A1 cells (J). (K) Validation of HDAC1-knockdown efficiency in A1, WA01 and iPS-IMR90-1 cells. (L) Functional effect of HDAC1-knockdown on G1-phase. Fold change in G1-cells is elevated upon HDAC1-knockdown in WA01 and iPS-IMR90-1 cells. Bars represent means ± SEM of three independent experiments. n-t - non-target siRNA control.