C1galt1 abrogation does not alter tight junction integrity or function.

<p>(A) Whole-cell protein lysates (15 µg) were resolved on SDS-PAGE and immunoblotted with antibodies against ZO-1, occludin and GAPDH, the latter serving as loading control. Comparable levels of junctional proteins were observed in C1galt1 shRNA keratinocytes as compared to controls. (B) Stratified cultures of corneal keratinocytes were fixed, permeabilized and stained for ZO-1. Confocal images corresponding to horizontal sections demonstrated ZO-1 distribution to the cell border in all cultures analyzed, consistent with tight junction localization in normal corneal epithelial cells <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036628#pone.0036628-Ryeom1" target="_blank">[39]</a>. Permeability measurements showed that TEER responses were not attenuated in C1galt1 shRNA cells as compared to controls. Data for each condition are reported as the mean of three wells in three independent experiments. Scale bar, 20 µm.</p

Targeted disruption of C1galt1 enhances nanoparticle uptake in human corneal keratinocytes.

<p>(A) HCLE-nt, scramble shRNA, and C1galt1 shRNA keratinocytes were incubated from the apical side with 100 nm FluoSpheres® suspensions (10¹⁰ nanoparticles/ml) for 3 h at 37°C, and subsequently examined by confocal microscopy. The position of cell nuclei was determined with PicoGreen. An increased number of nanospheres was observed in C1galt1 shRNA cells as compared to controls. When experiments were carried out at 4°C, a condition that blocks active transport processes, the internalization of nanospheres was dramatically reduced. Scale bar, 20 µm. (B) Examination of Z-stacked images showed that the nanospheres localized predominantly to the cytoplasm in C1galt1 shRNA cells incubated at 37°C. Scale bar, 20 µm. (C) Quantitative analyses of nanoparticle internalization were carried out by fluorometry of cell lysates. Nanoparticle internalization was significantly higher in C1galt1 cells as compared to controls. The uptake was dependent on the temperature and was impaired by treatment with the metabolic inhibitor sodium azide. Values are normalized to percent total uptake in HCLE-nt cells. Data for each condition are reported as the mean of three wells in three independent experiments. **<i>P</i><0.001 compared with scramble shRNA group at 37°C and ††<i>P</i><0.001 compared with C1galt1 shRNA group at 37°C.</p

C1galt1-deficient human corneal keratinocytes display plasma membrane invaginations on the apical surface.

<p>(A) Scheme for mucin-type O-glycan biosynthesis. The core 1 β,3-galactosyltransferase (C1galt1 or T-synthase) is a key branchpoint enzyme that directs the synthesis of core 1 (T-antigen), the precursor structure for many extended mucin-type O-glycans in a wide variety of glycoproteins. (B) Electron micrograph at low magnification of an ultrathin section (60 to 90-Å) of C1galt1 keratinocytes grown on Transwell® inserts demonstrating cell stratification and the presence of flattened epithelial cells on the apical layer (top) Scale bar, 10 µm. Membrane invaginations (arrowheads, inset) can be observed occasionally on the apical portion of the plasma membrane in apical cells of C1galt1 keratinocytes (middle), more frequently than in scramble control (bottom). Scale bar, 200 nm.</p

Targeted disruption of C1galt1 enhances endocytosis of biotinylated cell surface protein in corneal keratinocytes.

<p>(A) Cell surface protein was labeled with biotin at 4°C and then allowed to internalize for 15 or 25 min at 37°C. Crude postnuclear supernatants were ultracentrifuged using 5–25% Optiprep gradients and analyzed by agarose gel electrophoresis. Fraction 1 contains the lightest membranes; fraction 16 contains the densest membranes. The position of the plasma membrane and endocytic vesicles in the gradient was determined by western blot using antibodies to MUC16 and human TfR, respectively. Biotinylated protein was detected using streptavidin-peroxidase as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036628#s4" target="_blank">Materials and Methods</a>. (B) Quantitative evaluation of biotin accumulation in Optiprep density gradient. The graph shows the ratio of biotinylated protein in C1galt1 shRNA and scramble shRNA cells as determined by densitometry. Band intensity in each fraction was normalized to percent total biotin loaded in each gradient. Data for each condition are reported as the mean of three independent experiments.</p

Clathrin-mediated endocytosis is a predominant pathway for nanoparticle internalization in C1galt1 shRNA cells.

<p>(A) For fluorometry assays, corneal keratinocytes were pre-incubated at 37°C in the presence of inhibitors of endocytosis. After 30 min, FluoSpheres® suspensions (10¹⁰ nanoparticles/ml) were added to the cells in the continuous presence of inhibitors and incubated for 3 h at 37°C. Sucrose was directly added to the suspension in these experiments. Nanoparticle uptake was quantified by fluorometry as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036628#s4" target="_blank">Materials and Methods</a>. Values are normalized to percent total uptake in HCLE-nt cells. Data for each condition are reported as the mean of three wells in three independent experiments. **<i>P</i><0.001 compared with scramble shRNA group and ††<i>P</i><0.001 compared with the C1galt1 shRNA group with no inhibitors. (B) Confocal microscope images of C1galt1 shRNA cells. For colocalization experiments, keratinocytes were incubated with FluoSpheres®suspensions (red) for 3 h at 37°C. The cells were then fixed, permeabilized and stained for clathrin heavy chain or EEA1 (green). Examination of Z-stacked images shows colocalization of nanospheres with clathrin (Pearson's coefficient: 0.939) or EEA1 (Pearson's coefficient: 0.885) in the cytoplasm (arrowheads). Scale bar, 10 µm.</p

Isolation of the MUC16 sheddase and its identification as ZmpC.

<p>MUC16 sheddase-enriched fractions were isolated first by DEAE chromatography, with concentrations of sodium chloride eluent shown in the figure on the right (<b>A</b>), then by size exclusion chromatography (<b>B</b>). A sample from fraction 9 (indicated by an arrow) in B was separated by SDS-PAGE and stained using GelCode Blue stain. The 3 bands observed at ∼180 kDa, ∼170 kDa and ∼50 kDa were analyzed by mass spectrometry. Numbers on the left of the gel indicate molecular weight standards in kDa. The Y axes of graphs in (A) and (B) represent the net intensities corresponding to MUC16 signal on western blots. Constitutive MUC16 ectodomain release was observed in fractions whose net intensities correspond to that of the control. The mechanism(s) associated with constitutive MUC16 ectodomain shedding remains unknown.</p

Rose bengal penetrance into HCLE cells increases after SP168 growth culture filtrate treatment.

<p>Brightfield micrographs were taken of HCLE cells stained with rose bengal dye following exposure to (<b>A</b>) medium only control or (<b>B</b>) ZmpC-containing growth culture filtrate obtained from strain SP168. Areas of cells with dye penetrance (p = 0.01, Mann-Whitney test, n = 55) were quantified using ImageJ software (<b>C</b>). Scale bar = 100 µm. These data indicate that the barrier efficiency of epithelial cells after exposure to ZmpC decreases due to loss of MUC16 ectodomain. Increased dye penetrance has been shown previously with siRNA knockdown of MUC16 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032418#pone.0032418-Blalock1" target="_blank">[3]</a>.</p

Apical cell surface abundance of MUC16 is reduced following treatment of epithelia with SP168 growth culture filtrate.

<p>Apical membrane levels of MUC16 were determined by cell surface biotinylation. <b>A</b>) Western blot analyses (using M11 antibody) of the amount of released and residual biotin-labeled surface MUC16 after exposure to medium only control or SP168 growth culture filtrate. <b>B</b>) Quantitative analyses of the western blot in (A) showing a reduction in the abundance of surface MUC16 following treatment with the SP168 culture filtrate (p<0.0001, Student's t-test, n = 3). Data for MUC16 surface abundance is expressed as a percentage of the total released plus surface MUC16 ± SEM.</p

Disruption of galectin-3 binding and multimerization impairs glycocalyx barrier function.

<p>(A) One-hour preincubation of stratified human corneal epithelial cell cultures with β-lactose—but not with the non-inhibitory controls of galectin binding, sucrose and maltose—resulted in a significant and transient increase in rose bengal uptake. (B) Incubation with rhGal3 after treatment with β-lactose allowed recovery of barrier function in corneal epithelial cells. On the other hand, addition of rhGal3C resulted in sustained rose bengal uptake by the cell culture. Representative images are shown in the left panel. Images were obtained using a 10× objective lens. All the experiments were performed in triplicate and represent the mean ±SD. ns, not significant, **P<0.01, ***P<0.001.</p

Abrogation of galectin-3 impairs barrier function in mouse corneas.

<p>Numerical scoring for the intensity of staining with rose bengal revealed a higher incidence of epithelial defects in corneas of galectin-3 null mice (Gal-3^−/−) as compared to wild-type (Gal-3^+/+) controls. Representative images are shown in the left panel. *P<0.05.</p