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

Toward Liquid Biopsy: Determination of the Humoral Immune Response in Cancer Patients Using HaloTag Fusion Protein-Modified Electrochemical Bioplatforms

Autoantibodies raised against tumor-associated antigens have shown high promise as clinical biomarkers for reliable diagnosis, prognosis, and therapy monitoring of cancer. An electrochemical disposable biosensor for the specific and sensitive determination of p53-specific autoantibodies has been developed for the first time in this work. This biosensor involves the use of magnetic microcarriers (MBs) modified with covalently immobilized HaloTag fusion p53 protein as solid supports for the selective capture of specific autoantibodies. After magnetic capture of the modified MBs onto screen-printed carbon working electrodes, the amperometric signal using the system hydroquinone/H₂O₂ was related to the levels of p53-autoantibodies in the sample. The biosensor was applied for the analysis of sera from 24 patients with high-risk of developing colorectal cancer and 6 from patients already diagnosed with colorectal (4) and ovarian (2) cancer. The developed biosensor was able to determine p53 autoantibodies with a sensitivity higher than that of a commercial standard ELISA using a just-in-time produced protein in a simpler protocol with less sample volume and easily miniaturized and cost-effective instrumentation