Organ biodistribution of intraperitoneally-administered sRAGE or MSA in mice.

<p>Organ biodistribution of mouse sRAGE or MSA (A) 1 hour, (B) 2 hours, (C) 4 hours, and (D) 12 hours after intraperitoneal injection of ∼1 µCi of tracer, corresponding to ∼0.6–1.4 µg of radiolabeled protein. Asterisks indicate statistically significant differences.</p

Kinetics of binding of extracellular matrix proteins in solution to solid-phase sRAGE.

a<p>Not determined due to undetectable specific binding.</p

Mouse and human sRAGE extinction coefficients.

a<p>Mouse membrane RAGE has a published sequence length of 402 (NP_031451.2).</p>b<p>Human membrane RAGE has a published sequence length of 404 (NP_001127.1).</p>c<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088259#pone.0088259-Pace1" target="_blank">[50]</a>.</p

Organ biodistribution of intratracheally-administered sRAGE or MSA in mice.

<p>Organ biodistribution of mouse sRAGE or MSA (A) 1 hour, (B) 2 hours, (C) 4 hours, and (D) 12 hours after intratracheal instillation of ∼1 µCi of tracer, corresponding to ∼0.6–1.4 µg of radiolabeled protein. Asterisks indicate statistically significant differences.</p

Clearance of intratracheally-administered sRAGE from mouse lung occurs at the alveolar-capillary interface.

<p>Representative light micrographs/autoradiographs recorded at 20× magnification of sections of lung from mice given mouse sRAGE (or MSA, shown for comparison) i.t. and sacrificed at the indicated time points thereafter. Lung from untreated mice serves as a control for background radiation.</p

Clearance of intratracheally-administered sRAGE or MSA from mouse lung is rapid and non-proteolytic.

<p>(A) Exogenous mouse sRAGE or MSA persistence in mouse lung as a function of time. (B) SDS-PAGE separation and gel autoradiography of lung homogenates from mice administered i.t. mouse sRAGE or MSA and sacrificed at the indicated time points thereafter.</p

RAGE binds with high affinity to collagen I, collagen IV, and laminin, but not to fibronectin.

<p>Soluble RAGE affinity for collagen I, collagen IV, laminin, and fibronectin was assessed by biolayer interferometry. Soluble RAGE was conjugated to an amine-reactive sensor and dipped into solutions containing collagen I (A), collagen IV (B), laminin (E), or fibronectin (F) for 1800 s, at which point dissociation was initiated by washing in PBS. Binding was measured as a deflection in the wavelength of light passing through the sensor; affinities were calculated by fitting the curves with analysis software. Reciprocal binding was studied with collagen I (C) and collagen IV (D) conjugated to the sensor and sRAGE in solution. Laminin and fibronectin could not be conjugated to the sensor.</p

Analysis of BALF specimens 21 days after treatment.

<p>(A) Both silica treated WT and RAGE KO (closed bars) had significantly more total cells per mL in the BALF compared to control treated mice (open bars). There was not a significant difference between WT and RAGE KO mice. Furthermore, the number of macrophages in both WT and RAGE KO BALF was not significantly increased over control treated mice (B). While the level of neutrophils increased significantly with silica treatment compared to controls for both strains (C), only RAGE KO mice had a significant increase in lymphocytes after silica treatment (D). (E) Protein concentration in the BALF also increased significantly with treatment, but there was no difference between the two strains. Data are means (±SEM) analyzed by 2-way ANOVA with a Bonferroni post-test. (<i>n = 6–7 per group</i>). * p<0.05 silica vs. control.</p

sRAGE and MSA used in biodistribution and clearance studies are very pure.

<p>(A) SDS-PAGE separation and Coomassie Brilliant Blue staining of ∼2.5 µg of MSA or mouse sRAGE preparations used for radiolabeling. (B) SDS-PAGE separation and gel autoradiography of ¹²⁵I-labeled MSA and mouse sRAGE.</p

Lung mechanics after silica injury.

<p>Changes in lung mechanics were similar in WT and RAGE KO mice. Closed bars represent silica treatment and open bars vehicle control. (A) While the airway resistance in silica treated WT mice was not significantly increased over control, the RAGE KO mice treated with silica had a significant increase. All other values (B–E) were significantly changed for both WT and RAGE KO; however there were no other significant differences between WT and RAGE KO. Data are means (±SEM) analyzed by 2-way ANOVA with a Bonferroni post-test. (<i>n = 5–6 per group</i>). * p<0.05 silica vs. control.</p