Mediators of Inflammation / 2019 / Article / Fig 4

Research Article

PARP-1 Is Critical for Recruitment of Dendritic Cells to the Lung in a Mouse Model of Asthma but Dispensable for Their Differentiation and Function

Figure 4

(a) Lung sections from OVA-sensitized and OVA-challenged WT or PARP-1−/− mice were subjected to immunohistochemistry with antibodies to mouse VCAM-1; bar: 50 μm. Immunoreactivity was assessed using the Image-Pro software. Results are of immunoreactivity signals expressed in arbitrary units; . (b) PARP-1−/− endothelial cells were transduced with an adenoviral vector encoding human PARP-1 or control virus. Protein extracts were subjected to immunoblot analysis with antibodies to PARP-1 or actin. (c) Cells from (b) were treated with 1 mg/ml LPS, 10 ng/ml TNF-α, or 10 ng/ml IL-1β for 4 h. Total RNA was then prepared, reverse-transcribed, and amplified by PCR with primer sets (Supplemental Table 1) specific to human PARP-1, mouse VCAM-1, mouse iNOS, or GAPDH. Amplicons were subjected to agarose electrophoresis. (d) Lung smooth muscle cells isolated from WT or PARP-1−/− mice were subjected to increasing concentrations (0.01-1000 ng/ml) of LPS for 4 h. Isolated RNA was then reverse-transcribed followed by PCR with primers to mouse VCAM-1 or β-actin. (e) WT or PARP-1−/− smooth muscle cells were treated with 100 ng/ml LPS mice and collected after 18 h. Protein extracts were subjected to immunoblot analysis with antibodies to VCAM-1 or actin. (f) PARP-1−/− smooth muscle cells were transduced with the aforementioned adenoviral vectors after which cells were treated with LPS and collected 18 h later. Protein extracts were subjected to immunoblot analysis with antibodies to PARP-1, VCAM-1, or actin. (g) Protein extracts from differentiated WT, PARP-1−/−, or olaparib-treated WT DCs were subjected to immunoblot analysis with antibodies to VCAM-1 or actin. For (e–g), signals were quantified and are shown as values under the respective blots.
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