Aggravated lung injury and coagulation in Sirt1 knock-out mice after PM_2.5 exposure. (A) To generate inducible Sirt1 knock-out mice, floxed Sirt1 mice containing a loxP-flanked Sirt1 exon 4 were bred with UBC-Cre-ERT2 mice. Tamoxifen-inducible and Cre-mediated recombination resulted in deletion of Sirt1 exon 4. Inducible knock-out mice were given tamoxifen by intraperitoneal injection. Ten days after tamoxifen treatment, spleen, lung, and liver were collected. Tissue homogenates were subjected to immunoblotting to confirm Sirt1 deletion. (B) Sirt1^−/− and Sirt1^+/+ littermate mice were exposed to PBS or PM_2.5 (100 μg) by intranasal instillation. Twenty-four hours later, lungs were perfused with PBS via the right ventricle to remove blood from the vascular bed of the lung. Lung images show that PM_2.5 exposure led to blood clot formation (arrows). Images are representative of 6 mice in each experimental group. (C) Histologic examination by H&E staining shows increased leukocyte infiltration and blood clot formation in the lungs of Sirt1^−/− mice after PM_2.5 exposure. Images are representative of 4 mice in each experimental group. Sirt1^+/+ and Sirt1^−/− littermates were exposed to PBS or PM_2.5 (100 μg) by intranasal instillation. Twenty-four hours later, BAL fluids were collected. (D) BAL albumin levels were assessed by ELISA. (E) Total cells and neutrophils in the BAL fluids were counted by flow cytometry. Data are presented as mean + SEM (n ≥ 7 mice/group; *P < .05).