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. 2016 Feb;54(2):241–249. doi: 10.1165/rcmb.2015-0079OC

Figure 1.

Figure 1.

Difference in gene expression and promoter methylation level of phosphodiesterase 4D (PDE4D) between nonasthmatic and asthmatic airway smooth muscle cells (ASMCs). (A) mRNA expression level of PDE4D in asthmatic ASMCs (five donors) relative to nonasthmatic ASMCs (five donors) assayed by quantitative polymerase chain reaction (qPCR). Values are mean ± SD. **P < 0.01. (B) Schematic diagram of CpG dinucleotide (CG) content (%) in the 5′ promoter region of PDE4D. In silico analysis identified the CpG islands (shaded in gray in the genomic DNA sequence) based on the CG content greater than 60% with an observed:expected ratio of 0.6 (MethPrimer). ATG, translational start site; TSS, transcription start site; UTR, untranslated region. PCR amplicon generated by bisulfite sequencing PCR (BSPCR) and methylation-specific qPCR (MSPCR) indicated by the regions bounded by arrows. (C) Methylation status of individual CpG site at the PDE4D promoter in nonasthmatic (three donors) and asthmatic ASMCs (three donors) was assayed by bisulfite sequencing. Unmethylated (open circles) or methylated (solid circles) CpGs are indicated. Each row of circles represents an individual clone sequenced. Three to four individual clones from each donor were picked for sequencing. Boxed area illustrates the specific CpG sites showing differential methylation in asthmatic ASMCs (P < 0.01).