Fig. 3

OPG activates pro-proliferative signalling and a disease-relevant transcriptome. Panel (a) Signalling Pathway Impact Analysis (SPIA) with each pathway represented by one dot. The pathways to the right of the red diagonal line are significant after Bonferroni correction of the global p-values obtained using Fisher’s methods from the combination of pPERT and pNDE values, the pathways to the right of the blue line are significant after FDR correction. (b) shows a heat map of significant differentially regulated genes after gene enrichment against PAH-associated genes in OPG stimulated PASMCs, (c) TaqMan validation of gene expression microarray, TaqMan expression data normalised using ΔΔCT with 18 s rRNA as the endogenous control gene. Panel (d) shows a heat map of cell cycle/CDK proteins significantly regulated by OPG at 10 and 60 min expressed as a ratio to unstimulated controls from the same time point from Kinex phospho-arrays identified, with (e) showing those specifically related to NF-κβ. f Western blot validation of Kinex array data in unstimulated (0.2% FCS, Un) or OPG-stimulated (50 ng ml⁻¹) PASMCs at 10 min (10) and 60 min (60) with relative band densities of phospho-ERK1/2, phospho-HSP27, phospho-mTOR, phospho-CDK4 and total CDK5 are shown by the bar graphs and representative western blot images shown above the graph. Heat maps show Z-ratio gene or protein expression. Bars represent mean with error bars showing the standard error of the mean, n = 3 for pooled triplicate samples (a, b), n = 12 (c), n = 4 (d, e), n = 5 (f) from three donors of PASMCs, dots represent experimental repeats. Bars from unstimulated cells are white, OPG stimulated blue. *p < 0.05, ** p < 0.01, *** p < 0.001 compared OPG-stimulated to unstimulated PASMCs using one-way ANOVA followed by Bonferroni’s multiple comparisons post hoc test. When there were only two groups, unpaired t-tests were used