Wnt3a activated STAT3 in ARPE-19 cells.

<p>(A) Nuclear localization of phospho-STAT3 is used as a marker of activated STAT3. Incubation of sub-confluent cells with Wnt3a induced transient translocation of pSTAT3 (red) into the nucleus (arrows) after 5 hr incubation, but not after 24 hr. The inset shows a higher magnification. Overlap of pSTAT3 with the DNA dye DAPI (blue) was used to demonstrate nuclear localization. Incubation of cells with control media showed only cytoplasmic phospho-STAT3. Nuclear β-catenin was used as an endogenous marker of Wnt signaling and is observed at both 5 and 24 hr time-points. The absence of primary antibody control (2° only) demonstrates specificity of the immunostaining (see also isotype control antibody in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046892#pone.0046892.s004" target="_blank">Figure S4</a>). Scale bar in the top 3 rows represents 50 µM; bottom row, 20 µM. (B) Quantification of nuclear phospho-STAT3 and nuclear β-catenin in cells treated with Wnt3a or control for 5 and 24 hr. The percent of total cells with nuclear proteins is shown (n = 3, *p<0.05; ***p<0.001). Wnt3a transiently increased the number of cells with nuclear phospho-STAT3, indicating that Wnt3a activates the STAT3 pathway. (C) Quantification of nuclear pSTAT3 and nuclear β-catenin in cells that were cultured as a polarized post-confluent monolayer, and treated with Wnt3a or control media. Wnt3a induced significant increases in pSTAT3 nuclear localization at both 5 and 24 hrs of treatment (n = 3, *p<0.01). The percent of total cells with nuclear proteins is shown.</p

Activation of Wnt signaling in the RPE cell line ARPE-19.

<p>(A) Cultured cells were co-transfected with the TOP-FLASH Wnt reporter plasmid and a plasmid containing β-galactosidase, and then incubated with Wnt3a or control conditioned media. Luciferase activity was normalized to β-galactosidase activity. Wnt3a induced a 32-fold increase in luciferase activity compared with control (n = 4, **p = 0.01). (B) Immunodetection of endogenous β-catenin was used to confirm Wnt signaling induction. Cells incubated in control conditioned media have predominantly cytoplasmic and membrane-associated β-catenin (red, arrowheads), indicating low basal levels of Wnt signaling. Stimulation with Wnt3a induced translocation of β-catenin into the nucleus (right, arrows), indicating activation of the Wnt pathway. Scale bar 50 µM.</p

Activation of the Wnt pathway protected ARPE-19 cells from oxidative stress.

<p>Cellular viability was significantly increased in the presence of Wnt3a conditioned media compared with control conditioned media when oxidative stress is induced with either 0.4 mM hydrogen peroxide (H₂O₂) (n = 4, *p<0.05) (B) or 0.4 mM paraquat (PQ) (n = 9, *p<0.05) (C), both for 24 hr. The viability of uninjured cells was also modestly increased by Wnt3a (n = 9, *p<0.05) (A). Cell viability was also significantly increased by Wnt3a when cells were cultured as polarized post-confluent monolayers on permeable membranes in the presence of 1.6 mM paraquat (n = 3, *p<0.05) (D). Viability was measured by Cell Titer Blue assay.</p

Reducing STAT3 expression eliminated Wnt3a-mediated protection against oxidative stress.

<p>(A–B) Efficient reduction of STAT3 expression by siRNA. The cells were transfected with siRNA and STAT3 expression was measured by (A) QPCR (n = 3) and (B) Western blotting (n = 2), and normalized to untransfected cells. *p<0.05. C) The cells were transfected with STAT3 siRNA or a control scrambled siRNA and then were treated with H₂O₂ (0.8 mM for 5 hr) with and without Wnt3a. Viability was measured with Cell Titer Blue and normalized to untreated cells (UT). The rescue effect by Wnt3a was eliminated in the presence of STAT3 siRNA but was observed in the presence of control siRNA (n = 3, *p<0.05).</p

Wnt3a increased levels of nuclear phospho-STAT3.

<p>Nuclear fractions were prepared from cells treated with Wnt3a or control for 5 hr (W 5 h, C 5 h), or 24 hr (W 24 h, C 24 h). Lipopolysaccharide (LPS, 100 ng/ml, 5 hr) was used as a positive control, and untreated cells (UT) as the negative control. The blots were probed with antibodies against pSTAT3, total STAT3 (tSTAT3), and the nuclear marker protein lamin A/C. The 5 hr Wnt3a treatment increased the pSTAT3/tSTAT3 ratio by 25% compared with control (n = 3, p = 0.043), confirming that Wnt3a activates STAT3 and induces translocation into the nucleus. Representative blots for each antibody are shown.</p

Polarity of A1AT trafficking across cultured pulmonary endothelial monolayers.

<p>(A) Fraction (%) of FITC-Dextran (Dex) of 20 kDa (light gray bars) or 250 kDa (dark gray bars) or of A1AT (100 µg/mL; black bars) that crossed confluent endothelial cell monolayers grown on 0.4 µm transwells. (B) Immunoblots of intracellular and basolaterally secreted A1AT from endothelial cells cultured on transwell inserts treated with exogenous A1AT (100 µg/mL; for up to 120 min). (C) Intracellular levels of A1AT quantified by densitometry after normalizing to the vinculin loading control. (D) Transcytosed levels of A1AT normalized to .5 ul of concentrated supernatant. Bars represent mean+SEM; *p = .05 vs A1AT 2 h; #p<.05 vs. A1AT 10 min; n = 3.</p

Effect of cigarette smoke extract exposure on A1AT transcytosis across cultured lung endothelial monolayers.

<p>(A) Immunoblot of A1AT from concentrated supernatants showing basolaterally secreted A1AT in endothelial cells co-treated with A1AT (100 µg/mL) and CS or AC extract (2.5%) for up to 120 min (representative blot of n = 3). (B,E) Transcytosed A1AT levels in concentrated supernatants. Bars represent mean+SEM; *p<.05 vs. respective AC; n = 3. (C) Immunoblots of intracellular and basolaterally secreted A1AT in endothelial cells treated with native (Nat) or polymerized (Polym, heated at 60°, 2 h) A1AT (100 µg/mL; Baxter Healthcare). (D) Intracellular levels of A1AT (52 kDa) quantified by densitometry and normalized to the vinculin loading control. n = 2.</p

A1AT trafficking across cultured pulmonary endothelial and epithelial bilayers.

<p>(A) Co-culture schematic showing pulmonary epithelial cells cultured on the bottom of the transwell membrane and pulmonary endothelial cells cultured on the top. Endothelial cells only were exposed to A1AT. (B) Phalloidin staining (top and epithelial cells) and brightfield microscopy (bottom) showing confluent monolayers of endothelial and epithelial cells seeded on transwell inserts. (C) Immunoblotting of A1AT in cell lysates showing intracellular presence of A1AT in endothelial and epithelial cells and immunoblot of secreted A1AT from concentrated bottom supernatant (representative blots of n = 3). Bands shown are from the same immunoblot. (D). Schematic showing A1AT treatment of the basolateral media or apical surface of NHBE cells differentiated at ALI. (E). Confocal microscopy of NHBE cells differentiated at ALI after 2 h incubation with fluorescently labeled A1AT (green, 20 µM) added to either the basolateral media (i) or apical surface (ii). Only the basolaterally applied A1AT was observed to enter the cells. Arrows indicate ciliated side of the epithelium (cilia stained in white, nuclei in blue). (F). Densitometric quantification of epithelial cell lysates and ASL collected at the indicated times after adding 20 µM of A1AT in either the basolateral (solid line, triangles) or the apical (solid line, circles) compartment from 3 different lung donors measured by Western blotting (mean+SEM; n = 3). Plot (dashed line, squares) showing the relative A1AT present in the ASL after the basolateral application experiment. All fold changes are relative to time 0 before the addition of A1AT. (G) Concentration of A1AT in ASL of ALI cultures after A1AT (20 µM) addition to the basolateral compartment. ASL was collected with 250 µl PBS washes. A1AT quantification was made by customized ELISA and corrected for wash dilution (n = 3 different lung donors). (H) Levels of intracellular and secreted A1AT measured by ELISA (n = 2) in NHBE cells treated with conditioned endothelial media (containing 43.4 nM endothelial-secreted A1AT, 2 h);. n = 1.</p

Intravital microscopy of A1AT trafficking across the lung microvascular circulation in the intact mouse.

<p>Selected frame from timelapse video of the pulmonary microcirculation following intravenous injections of rhodamine-labeled rat albumin (red) and airspaces (dark) showing no AF488-A1AT (green) prior to injection (pre-A1AT). Note that following AF488-A1AT intravenous administration there is circulating protein (orange microcirculation) as well as A1AT protein in the airspaces (green punctate signal), without evidence of lung edema (no red extravasation), suggesting active transcytosis from the circulation at 10 min and 50 min post injection.</p

Secretory pathways leading to A1AT transcytosis across cultured pulmonary endothelial monolayers.

<p>(A–D) Immunoblot showing effect of inhibition of classical secretory pathway with tunicamycin (A–C; at the indicated doses; 18 h) or brefeldin A (D; 1 µg/mL, 60 min) on intracellular A1AT (A, B, D) and secretion of A1AT (A, C) detected by Western blotting and quantified by densitometry, using vinculin as loading control. Secreted A1AT was measured in equal volumes of supernatants (which were concentrated 25-fold). (E) Pre-inhibition of the classical secretory pathway with tunicamycin (1 h) enhances A1AT (3 h) basolateral secretion, measured by Western blotting of concentrated supernatants, suggesting the utilization of a non-classical secretory pathway. Bands are from the same immunoblot. (F) Alternative secretion of A1AT (100 µg/mL, 2 h) by endothelial cells via microparticles release, as detected by Western blotting of A1AT in endothelial microparticles isolated from supernatants via ultracentrifugation (representative blot of n = 3). (G) Time course of basolateral EMP release from endothelial cells treated with A1AT (100 µg/mL). Bars represent mean+SEM; *p<.05, #p = .07 vs. control; n = 3.</p