Figure 3.

Inhibition of DNA methyltransferase promotes DSP (desmoplakin)/Dsp expression on soft matrix. (A) Human (A549) and (B) mouse (primary mouse type II alveolar epithelial cells) lung epithelial cells were cultured on soft and stiff matrix in the presence or absence of 5 mM 5-aza-2′-deoxycytidine (5-aza-CdR) for 48 hours. Levels of DSP/Dsp mRNA and protein were determined by quantitative PCR (qPCR) and immunoblot analysis. GAPDH/Gapdh were used as reference/loading controls. DMSO is a solvent of 5-aza-CdR. (C and D) DNA methylation profiles in the conserved 560-bp regulatory region were determined by sodium bisulfite sequencing. Ten randomly picked DNA clones were sequenced for each condition. Solid circles indicate methylated CpG islands. Open circles indicate unmethylated CpG islands. Results are mean ± SD of three or four independent experiments, each qPCR reaction performed in triplicate. *P < 0.05. AP-2 = activator protein-2; C/EBPβ = CCAAT/enhancer-binding protein β; dist = distal; EGR1 = early growth response protein 1; ETF = EGFR [epidermal growth factor receptor]-specific transcription factor; prox = proximal.