Figure 4.

EGR1 (early growth response protein 1) transactivation is associated with stiff matrix–induced DSP (desmoplakin) overexpression. (A) Schematic depicts the wild-type (open shapes) and mutated (solid shapes) 560-bp regulatory sequences. Promoter activity was determined by a luciferase-based assay. (B) The bindings of EGR1 to the distal and proximal EGR1 binding sites (EBS) under soft versus stiff matrix conditions were measured by quantitative chromatin immunoprecipitation. (C) The bindings of nuclear EGR1 from human alveolar epithelial adenocarcinoma cells with unmethylated or methylated DSP promoter probes were determined by electrophoretic mobility shift assay. (D) Levels of EGR1 in the whole-cell lysates, cytoplasmic fraction, and nuclear fraction were determined by immunoblot. GAPDH and lamin B were used as loading controls for the cytoplasmic and nuclear proteins, respectively. (E) Knockdown of EGR1 by siRNA was confirmed by immunoblot. Effects of EGR1 knockdown on stiff matrix–induced DSP expression were determined by immunoblot. Results are mean ± SD of at least three or five independent experiments. *P < 0.05. Ab = antibody; C/EBPβ = CCAAT/enhancer-binding protein β; Ctrl = control; dist = distal; ETF = EGFR [epidermal growth factor receptor]-specific transcription factor; Met = methylated; prox = proximal.