Fig. 4
Phosphoproteomics elucidate downstream effectors of PI3Kα in endothelial cells. a Schematic illustration of the untargeted label-free mass spectrometry analysis. The study was conducted in Pik3caflox/flox (control) and Pdgfb-iCre;Pik3caKD/flox (Pik3caKD/iΔEC) mouse lung endothelial cells under exponential growing conditions upon preincubation with vehicle (EtOH) or 4-OHT for the indicated time points. The vehicle condition for analysis of the heterozygous inactivation of PI3Kα (Pik3caKD/flox without induction of CRE activity) was included as a further control and four different mice were analysed in each condition (a total of 24 samples). b Volcano plots exhibiting changes in phosphopeptides across genotypes. The Y axis represent the negative log10 of P value and the X axis shows the log2 of the fold change between control and Pik3caKD/iΔEC endothelial cells treated with vehicle (EtOH) for 24 h, 4-OHT for 24 h or 4-OHT for 96 h. Red and yellow dots represent significantly regulated phosphopeptides (P < 0.01 and P < 0.05 respectively) with a fold-change higher than 0.8 or lower than −0.8. c Venn diagram showing the number and percentage of phosphopeptides which are significantly upregulated or downregulated between experimental groups. Number and percentage of overlapping phosphopeptides between groups are also shown. d Heatmap indicating fold-changes in the phosphorylation of proteins related to the cytoskeleton. Phosphopeptides identified to be down- or upregulated in Pik3caKD/iΔEC vs. Ctrl are shown in blue and red, respectively across EtOH and 4-OHT treatments. Values shown represent mean fold-change over Ctrl. e Western blot validation of pS445 MYPT1 in mouse lung endothelial cells and HEK-293 cells upon genetic and pharmacological inhibition of PI3Kα. Control and Pik3caKD/iΔEC endothelial cells were treated with 4-OHT for 72 h, re-platted for 24 h and subjected to immunoblotting. Wild-type endothelial cells and HEK-293 cells were treated with vehicle or GDC-0326 for 48 h and subjected to immunoblotting. Quantification of at least three independent experiments is shown in Supplementary Figure 8
