Journal: Nature Communications
Article Title: A therapeutic antibody targeting osteoprotegerin attenuates severe experimental pulmonary arterial hypertension
doi: 10.1038/s41467-019-13139-9
Figure Lengend Snippet: 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
Article Snippet: Cells were then stimulated with 0.2% (v/v) FBS (negative), rhOPG (50 ng ml −1 ) and PDGF (20 ng ml −1 ) for 10 and 60 min. Phosphorylation targets were identified from protein lysates by Kinex antibody microarray (Kinexus, Vancouver, Canada).
Techniques: Expressing, Microarray, Western Blot