Poly(I:C) induces ET-1 gene through the PKR-dependent activation of the eIF2α/ATF4 pathway.

<p>80% confluency, HDMECs were treated with 20 nM PKR siRNA (siSCR) with or without Poly(I:C) treatment (a and b). Total RNA was extracted and mRNA level of PKR (a) and PPET1 (b) were quantified by quantitative RT-PCR. Expression of the housekeeping gene β-actin served as an internal positive control in each assay performed. After measurement of the relative fluorescence intensity for each sample, the amount of each mRNA transcript was expressed as a threshold cycle value. 20 µg of nuclear extract were separated via 15% SDS-PAGE for ATF4 (c), 20 µg of total cell lysate were separated via 10% SDS-PAGE for peIF2α/eIF2α (d), then transferred to a nitrocellulose membrane. The blots were probed overnight with primary Abs at 4°C. As a control for equal protein loading, membranes were stripped and reprobed for Lamin A/C or β-actin. Representative blots of at least three experiments are shown. (e) Expression of ATF4 by real-time PCR analysis of skin mRNA from C57B1/6 WT (n = 10), C57B1/6 IFNAR1−/− (n = 8) and C57B1/6 TRIF/TICAM −/− (n = 6) mice 1 week after subcutaneous insertion of osmotic pumps containing Poly(I:C). Fold-change shown in the graphs is normalized to mRNA expression by one of the control mice. *<i>p</i> = 0.05; **<i>p</i> = 0.001 (f) ATF4 protein expression in dermal biopsies obtained from C57Bl/6 WT mice 1 week after subcutaneous insertion of osmotic pumps containing Poly(I:C) or PBS (as control), and processed for immunohistochemistry as described under Methods. Representative images of microvessels from PBS and Poly(I:C) skin is shown. Bar: 20 µm, 10 µm.