Immunohistochemistry.

<p>Lung endothelial cells are labeled with anti-ERG antibody (green) and proliferating cells are marked with anti-Ki67 antibody (red). Double-stained Ki67-positive endothelial cells appear yellow (arrows). Representative sections of control and VEGF-treated lungs harvested on POD 2 (A) and 4 (B) are shown at 20X magnification. Topical VEGF treatment significantly increases endothelial proliferation on both POD 2 and 4 (C).</p

Lung tissue protein expression analyses.

<p>ELISA reveals increased VEGF levels in VEGF-treated lungs (A) on POD 4. However, quantitative polymerase chain reactions (qPCR) show no difference in mRNA transcript levels of VEGF, VEGFR1, or VEGFR2 between the two groups (B). Immunoblot demonstrates an increase in the levels of P-VEGFR2, VEGFR2, P-EGFR, EGFR, and heparin-binding EGF-like growth factor (HB-EGF) with VEGF treatment (C-D). Data are expressed as mean ± SE.</p

Morphometric analyses.

<p>Lungs of VEGF-treated mice demonstrate increased parenchymal volume (A) and a trend toward higher alveolar volume (B). Septal surface area (C) was higher in the VEGF group but there was no difference in mean septal thickness (D). VEGF-treated mice had increased total alveolar count (E). Data are expressed as mean ± SE.</p

Lung volume and functional assessments.

<p>Nasal instillation resulted in less systemic absorption of VEGF compared to intraperitoneal injection as measured by enzyme-linked immunosorbent assay (ELISA) (A). VEGF-treated mice display higher post-euthanasia lung volume on post-operative day (POD) 4 and 10 (B). There is a trend toward increased total lung capacity (C) and pulmonary compliance (D) with VEGF treatment on POD 4. Although not reaching statistical significance, mice in the VEGF group performed better on post-treadmill rest time (E), walking distance (F), basic movement count (G), and fine movement count (H) than the control mice on POD 10. Data are expressed as mean ± standard error of the mean (SE).</p

Co-culture assays of human bronchial epithelial cells (HBEC) and human lung microvascular endothelial cells (HMVEC-L).

<p>VEGF₁₆₅ at 10 ng/mL activated HMVEC-L (A). HMVEC-L treated with VEGF₁₆₅ show upregulation of proteases such as cathepsin B and V, matrix metalloprotease (MMP)-7, CD10, and kallikrein 13 (B) as well as a more than 6-fold increase in HB-EGF concentration in the conditioned medium (C). HBEC treated directly with VEGF show no increase in proliferation (D). However, in a co-culture model with HMVEC-L (E), proliferation increases when HBEC is co-cultured with VEGF-treated HMVEC-L (F). When an HB-EGF neutralizing antibody is added to co-cultured HBEC (G), proliferation decreases at higher concentrations of the antibody (H). Activation of EGFR on co-cultured HBEC is confirmed to decrease in the presence of an HB-EGF neutralizing antibody (I-J).</p

A purified DDB2 protein complex can be used to detect UV-induced DNA damage.

<p>(<b>A</b>) Experimental strategy to prepare the DDB2 proteo-probe. (<b>B</b>) Signal obtained by hybridization of the DDB2 proteo-probe onto fibroblasts with or without damaging treatments. Hybridized DDB2 proteo-probe is revealed by anti-HA immunofluorescence. Nuclei are visualized by DAPI staining. Nuclei are delineated based on DAPI staining and using CellProfiler <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085896#pone.0085896-Carpenter1" target="_blank">[26]</a>.</p

The decrease of DDB2 proteo-probe and 6-4 PP signals over time are nearly identical.

<p>(<b>A</b>) Typical signals after UV damage observed <i>in situ</i> with the DDB2 proteo-probe, an anti-CPD antibody, or an anti-(6-4)PP antibody. Nuclei are delineated based on DAPI staining and using CellProfiler. (<b>B</b>) The DDB2 proteo-probe signal decreases exponentially with time. Average signal per nucleus normalized to signal at 5 minutes. Red dashed curve: one phase exponential decay fit calculated with a non-linear least square method (R² = 0.86). (<b>C</b>) The anti-(6-4)PP signal decreases exponentially with time. Average signal per nucleus normalized to signal at 5 minutes. Blue dashed curve: one phase exponential decay fit calculated with a non-linear least square method (R² = 0.83). (<b>D</b>) The anti-CPD signal remains constant over a two hour period. Average signal per nucleus normalized to signal at 5 minutes. Black dashed line: linear fit on the α-CPD signal (R² = 0.18). (<b>B</b>), (<b>C</b>), and (<b>D</b>): cells were irradiated with UV-C (10 J/m²). The average of three replicas is shown. Each replica represents an average of at least 60 cells. Error bars: s.e.m. (<b>E</b>) A single one phase exponential decay model summarizes the kinetic of (6-4)PPs removal <i>in situ</i>. The single model is based on the decay fits obtained with DDB2 proteo-probe and anti-(6-4)PP data. The grey band represents the area enclosing the true decay curve with 99% confidence. The dotted line indicates the predicted half-life (<i>t</i>_1/2) of (6-4)PPs <i>in situ</i> after UV irradiation.</p

The DDB2 proteo-probe recognizes 6-4-photoproducts <i>in vitro</i>.

<p>(<b>A</b>) The DDB2 proteo-probe signal increases linearly with fluence (J/m²). Fibroblasts were irradiated with different doses of UV-C. Each point is an average of three replicas. Each replica represents an average of at least 60 cells. Dashed line: linear fit (R² = 0.94). Error bars: s.e.m. (<b>B</b>) The DDB2 proteo-probe signal is DNA-dependent. Fibroblasts were irradiated with UV-C (10 J/m²), and untreated or treated with DNase. Nuclei are visualized by DAPI staining. (<b>C</b>) The DDB2 proteo-probe signal can be competed with UV-treated plasmid DNA. Fibroblasts and plasmid DNA were irradiated with UV-C (10 J/m² and 300 J/m², respectively). The DDB2 proteo-probe was incubated with plasmid DNA prior to hybridization onto irradiated fibroblasts. Dashed line: no plasmid control proteo-probe signal level. Each point is an average of three replicas. Each replica represents an average of at least 400 cells. Error bars: s.e.m. (<b>D</b>) The DDB2 proteo-probe binds preferentially to 6-4-photoproducts [(6-4)PP] over cyclobutane pyrimidine dimers (CPD). The DDB2 proteo-probe was immobilized on agarose beads, and incubated with the DNA restriction fragments of a plasmid containing, or not, a unique lesion [(6-4)PP or CPD]. The average ratio of the amount of lesion-containing over lesion-free DNA fragments bound to the proteo-probe is shown (<i>n</i> = 3). Error bars: s.e.m.</p