Mediators of Inflammation / 2019 / Article / Fig 2

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 2

Bone marrow cells isolated from WT or PARP-1−/− mice were cultured in complete medium with 20 ng/ml GM-CSF. WT cells were treated with GM-CSF in the presence of 1 μM olaparib (AZD2281) or vehicle. The drug was added with every media change. On day 8, nonadherent bone marrow-derived DCs were phenotyped by flow cytometry with the fluorescently labeled antibodies CD11c-APC, CD11b-PE-Cy™7, MHCII-PerCP-Cy™5.5, CD40-BV421, CD80-FITC, and CD86-BV711. (a) The number of CD11b+/CD11c+/MHCIIhigh DCs per ml of culture medium in the different experimental groups. (b) Percent of CD11b+/CD11c+/MHCIIhigh DCs in the different experimental groups. (c) Representative FACS dot plots of the groups from (b). Percent of CD11b+/CD11c+/MHCIIhigh DCs expressing CD80 (d), CD86 (e), or CD40 (f). (g–h) Sorted CD11c+/CD11b+/MHCIIhigh cells from the different experimental groups were pulsed with OVA 323-339 peptide or vehicle overnight, washed, and then cocultured with CFSE-stained CD4+ T cells from OTII mice for four days. A portion of CD4+ T cells were stimulated with a combination of anti-CD3 anti-CD28 antibodies as a positive control. Proliferation was assessed by flow cytometry. For (a, b) and (d–h), the results are expressed as ; ; ; ; . (h) Recombinant PARP-1 was incubated with NAD in the presence or absence of olaparib and activated with double-stranded DNA breaks (DSB) for 30 min. Reactions were stopped by SDS sample buffer and subjected to immunoblot analysis with antibodies to poly(ADP-ribose) (PAR). The smear-like band is typical in poly(ADP-ribosyl)ation reactions showing PARP-1 with different levels of automodification.

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