Figure 4.
cGMP (cyclic guanosine monophosphate)-protein kinase G (PKG)-signaling pathway is down-regulated in aortas with vascular smooth muscle (VSM) bcl11b (B-cell leukemia 11b) deletion. A, Venn diagram of genes differentially up- and down-regulated in aortas of tamoxifen-inducible VSM-specific Bcl11b null mice (BSMKO; n=5) compared with wild-type (WT; n=5) mice assessed by RNA sequencing. B, Volcano plot of differentially up- (red) and down-(blue) regulated genes in aortas of BSMKO (n=5) compared with WT (n=5) mice. The horizontal line indicates a threshold of P=0.05. For a list of the top 40 differentially regulated genes see Figure IV in the Data Supplement. C, List of genes within the cGMP-PKG signaling pathway, the most significantly regulated signaling pathway in BSMKO aortas after Database for Annotation, Visualization and Integrated Discovery (DAVID) network analysis of differentially expressed genes (FDR, q=0.0094). For quantitation of individual genes see Figure VI in the Data Supplement. D, Quantitative RT-polymerase chain reaction (PCR) for guanylyl cyclase isoforms Gucy1a1, Gucy1a2, and Gucy1b1 in mRNA extracts from WT and BSMKO VSM cells; n=4 replicate experiments. Data expressed as fold change vs WT. *P=2.8×10−2; †P=1.6×10−2; ‡P=2.8×10−2 by Mann-Whitney nonparametric test. E, Representative Western Blot for PKG1 (PKG, isoform 1) protein levels in VSM cells from WT and BSMKO mice. GAPDH used as loading control. Quantitation of 4 replicate experiments in graph. *P=3.0×10−2 by Mann-Whitney nonparametric test. F, Representative Western Blot for PKG1 in mouse VSM cells treated with a scrambled siRNA (control) or a validated Bcl11b siRNA. Quantitation of 3 replicate experiments on 3 different mouse VSM cell lines in graph. β-tubulin used as loading control. *P=4.0×10−2 by unpaired t test.