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1.
Figure 5

Figure 5. Both normal and asthmatic individuals have shown a similar expression of 15-LOX-1 in bronchial epithelia.. From: 12/15-lipoxygenase expressed in non-epithelial cells causes airway epithelial injury in asthma.

(A), (B) The expression of 15-LOX-1 in lung sections of healthy controls and patients with asthma. Brown color indicates positive expression of 15-LOX-1; all images in A are shown at 20× magnification and in B at 100× magnifications (n = 6 in mice and 3 to 5 in humans).

Ulaganathan Mabalirajan, et al. Sci Rep. 2013;3:1540.
2.
Figure 4

Figure 4. Various inflammatory cells but not bronchial epithelia have shown the 12/15-LOX expression in mice.. From: 12/15-lipoxygenase expressed in non-epithelial cells causes airway epithelial injury in asthma.

(A) The expression of 12/15-LOX in lungs of mice that are PBS sensitized and challenged (SHAM) and OVA sensitized and challenged (OVA). Brown color indicates positive expression of 12/15-LOX; Images are shown at 20× and 100× magnifications. (B) Flow cytometric co-immunostaining of 12-LOX with markers of various inflammatory cells such as Gr-1, CD11b, CD11c, CD3, Siglec F, and F4/80 in single-cell suspensions of lung tissue from SHAM and OVA mice.

Ulaganathan Mabalirajan, et al. Sci Rep. 2013;3:1540.
3.
Figure 7

Figure 7. IL-13-mediated epithelial injury is reduced by 12/15-LOX deficiency.. From: 12/15-lipoxygenase expressed in non-epithelial cells causes airway epithelial injury in asthma.

Murine rIL-13 was administered as shown in Materials and Methods. Lung mitochondria and cytosols were examined for activities of complex I & IV, mitochondrial membrane potential and cytochrome C (A–D). TUNEL apoptotic assay (E) and the percentage of TUNEL-positive bronchial epithelium (F). Arrows indicated the apoptotic bronchial epithelia. (G) Ultrastructural changes of bronchial epithelial mitochondria were analyzed by TEM in Balb/c mice. Lung mitochondria and cytosols were examined for activities of complex I and cytochrome C (H–I) in 12/15-LOX Knockout mice. Results are shown as quantile box plots. The line across the middle of the quantile box is the median; the ends of the box are 25th and 75th quantiles and the whiskers extending from either end of the box are the 10% and 90% quantiles.

Ulaganathan Mabalirajan, et al. Sci Rep. 2013;3:1540.
4.
Figure 3

Figure 3. 12/15-LOX deficiency reduces epithelial injury in murine asthma.. From: 12/15-lipoxygenase expressed in non-epithelial cells causes airway epithelial injury in asthma.

Mice were grouped (n = 5–6 mice each group) and named as per status of sensitization/challenge/treatment. Complex IV activity (A), Complex I activity (B), Mitochondrial membrane potential (C), cytochrome C levels (D), TUNEL assay (E), caspase 3 activity of lungs (F) and TEM images of lung sections (G) in Balb/c mice. Cytochrome C levels in 12/15-LOX knockout C57BL/6 mice (H). MITO, mitochondria. CYTO, cytosol. Red arrows indicate the normal mitochondria with well developed cristae and Red arrowheads indicate the abnormal mitochondria with loss of cristae and swelling. Results are shown as quantile box plots. The line across the middle of the quantile box is the median; the ends of the box are 25th and 75th quantiles and the whiskers extending from either end of the box are the 10% and 90% quantiles.

Ulaganathan Mabalirajan, et al. Sci Rep. 2013;3:1540.
5.
Figure 1

Figure 1. 12/15-LOX overexpression in naïve mouse leads to asthma features.. From: 12/15-lipoxygenase expressed in non-epithelial cells causes airway epithelial injury in asthma.

(A) Healthy Balb/c mice were administered either 12/15-LOX plasmid (12/15-LOX OE) or control plasmid (Control). (B–D) Western blot and ELISA to determine 12/15-LOX expression, levels of 13-S-HODE and 12-S-HETE in lung cytosol. (E) Airway resistance in response to increasing concentrations of methacholine as the percent baseline airway resistance assuming saline aerosol-derived values as baseline (n = 5–6 each group) was determined. Photomicrographs of bronchovascular regions stained with hematoxylin and eosin (F), periodic acid-Schiff (G), and Masson trichrome (H) stains of lung sections. I) Inflammation score was determined in lung sections. Data are representative of two independent experiments. Results are shown as quantile box plots for (13-S-HODE and 12-S-HETE) or as mean ± s.e.m (airway resistance and inflammation score). The line across the middle of the quantile box is the median; the ends of the box are 25th and 75th quantiles and the whiskers extending from either end of the box are the 10% and 90% quantiles. Significance is determined with unpaired Student t test or Mann-Whitney test (***P < 0.05).

Ulaganathan Mabalirajan, et al. Sci Rep. 2013;3:1540.
6.
Figure 2

Figure 2. 12/15-LOX overexpression in naïve mouse leads to bronchial epithelial injury.. From: 12/15-lipoxygenase expressed in non-epithelial cells causes airway epithelial injury in asthma.

Mitochondrial complex IV activity (A), mitochondrial membrane potential (B), cytosolic cytochrome C levels (C), and cytosolic caspase 3 activity in lung cytosol (D). MITO, mitochondria. Photomicrographs of Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) stained lung sections (E) and the percentage of TUNEL-positive bronchial epithelium (F). (G) TEM images of lung sections. Red arrows indicate the normal mitochondria with well developed cristae and Red arrowheads indicate the abnormal mitochondria with loss of cristae and swelling. Results are shown as quantile box plots. The line across the middle of the quantile box is the median; the ends of the box are 25th and 75th quantiles and the whiskers extending from either end of the box are the 10% and 90% quantiles.

Ulaganathan Mabalirajan, et al. Sci Rep. 2013;3:1540.
7.
Figure 6

Figure 6. 12/15-LOX metabolites induce intrinsic apoptosis in bronchial epithelia.. From: 12/15-lipoxygenase expressed in non-epithelial cells causes airway epithelial injury in asthma.

(A) The concentration of 12-S-HETE was estimated in serum, sputum supernatant, and BAL fluid supernatant of healthy controls (Controls) or patients with atopic asthma (Atopic asthmatics). Results shown as mean ± s.e.m., significance was determined with unpaired Student's t test (*P < 0.0001). N = 40, 5, and 3 for serum, sputum, and BAL fluid supernatant, respectively, for controls and N = 80, 5, and 3 for serum, sputum, and BAL fluid supernatant, respectively, for atopic asthmatics. (B) The concentration of 13-S-HODE was estimated both in cytosol and culture supernatant of bronchial epithelium at IL-13 induction at the times indicated. (C) The concentration of 13-S-HODE was estimated both in cytosol and culture supernatant of bronchial epithelium with 48 hrs induction of either IL-13 alone or combination with IL-4 or TNF-α. (D) The concentration of 13-S-HODE was estimated both in culture supernatants of macrophages or fibroblasts at IL-13 induction for 24 hrs. Bronchial epithelia were incubated with 12/15-LOX metabolites rich culture supernatants of macrophages that were induced with 25 ng/ml IL-13 for 24 hrs and JC-1 staining (E) was performed and cytosolic cytochrome C levels (F) were estimated in those bronchial epithelia along with induction of different concentrations of 12-S-HETE. Carbonyl cyanide m-chlorophenylhydrazone (CCCP) treated bronchial epithelia stained with JC-1 was act as a positive control. MacroØ C.S., macrophage culture supernatant; Data are representative of two independent experiments. Results are shown as quantile box plots except 13-S-HODE and cytochrome C which were expressed in mean ± s.e.m. The line across the middle of the quantile box is the median; the ends of the box are 25th and 75th quantiles and the whiskers extending from either end of the box are the 10% and 90% quantiles. Significance is determined with unpaired Student t test (***P < 0.05).

Ulaganathan Mabalirajan, et al. Sci Rep. 2013;3:1540.

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