Multilabel immunohistochemical analysis of Muc2 in the mouse colon.

<p>(<b>A</b>) AB–PAS staining of a paraffin-embedded mouse colon section showing the inner (i) and the outer (o) mucus blankets. The inner layer exhibits a horizontal stratified arrangement while the outer layer, which is more difficult to preserve and is partly detached, is thicker than the inner layer and shows an alternation of horizontal laminated multilayered structures and loose curl-like structures. g: goblet cell. (<b>B</b>) and (<b>C</b>) Immunofluorescence analysis of Muc2 and UEA1. Only the inner mucus blanket is preserved in C. Muc2 was visualized using an anti-MUC2 antibody (green) that recognizes both human and mouse Muc2 and UEA1 lectin (red) that recognizes H-type 2 epitopes. Goblet cells (arrows) discharge Muc2 molecules that do or do not carry H type 2 epitopes demonstrating that UEA1-negative mucin does not result from degradation of UEA1-positive Muc2 polymers by bacteria. All UEA1-positive goblet cells (red) are Muc2 positive while not all Muc2 positive goblet cells are UEA1 positive. The high power in C shows exocytosis of Muc2 with no H type 2 epitopes (green) from a goblet cell close to another goblet cell that is discharging Muc2 carrying H type 2 epitopes (red) into the lumen. The inner mucus blanket is made up of multiple unmixed horizontal layers of mucin flanked by the most fucosylated layers. Bars  =  50 µm. Lu: lumen.</p

Alternation of goblet cell types in the mouse ileum producing unmixed Muc2 glycoforms.

<p>(<b>A</b>) AB–PAS-stained section from a mouse ileum showing the glycocalyx (purple) and the mucus gel between villi and at the top of the villi. Gx: glycocalyx; g: goblet cell. (<b>B–C</b>) Ileum section stained as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018761#pone-0018761-g001" target="_blank">Fig. 1</a>. Nonfucosylated and fucosylated Muc2 mucin polymers secreted along villi (high power Ba) and within crypts (high power Bb) form a long filamentous sticky rope-like structure made of unmixed Muc2 glycoforms. Arrows outline the diversity of goblet cells depending of the fucosylation level of Muc2. (<b>D</b>) Colocalisation of Muc2 (green) and MAA (red) which recognized the sialic acid α-2,3-galactose epitope. No goblet cell subpopulations were observed. Bars  =  50 µm. Lu: lumen.</p

Impact of Mini5B expression on the dissemination of MCF7 cell xenografts in immunodeficient mice.

<p>(<b>A</b>) Immunofluorescence analysis was performed on paraffin-embedded sections of tumors from mice injected with the Ires-Luc clone, the Mini5B-Luc CYS shRNA clone, the Mini5B-Luc clone and the Mini5B-Luc empty shRNA clone and stained with anti-MUC5B antibody. Mini5B was expressed and secreted by tumoral cells in tumors of Mini5B-Luc group and Mini5B-Luc empty shRNA. (<b>B</b>) Serial sections of livers of mice injected with the Ires–Luc clone and stained with HE and anti-PCNA antibody, and serial sections of lungs of mice injected with the Mini5B–Luc clone and stained with HE and anti-PCNA antibody. Metastases in the liver and the lung were visualized. Metastatic cells were PCNA positive. (<b>C</b>) Histological analysis of a macrometastasis in lung of a mouse injected with the Mini5B–Luc clone using HE staining and E-cadherin immunostaining (serial sections). Mt; metastasis. Nuclei were counterstained with Hoechst 33258. Scale bar 50 µm.</p

Depletion of CGC is restored by IL13.

<p>The beanplots show that daily topical rIL13 application in the DES model significantly restored (<i>P</i><0.0001, one-sided Wilcoxon-Mann-Whitney test) the GFP⁺ CGCc density (n = 44, PBS-control mice (blue); n = 39, rIL13-treated mice (purple)). Mice from the control group received BAK for 10 days in one eye (PBS in the other) followed by 4 days of PBS in both eyes. Mice from the rIL13 group received BAK in both eyes for 10 days followed by rIL13 in one eye and PBS in the other for 4 days. The percentage of GFP⁺ CGCc in the treated eye vs. the non-treated eye was calculated for each mouse from pCLE movies. Sample median is shown as horizontal black bars.</p

CGC counting (7 mice, 3 sections/mouse) in left (PBS) and right eye (BAK).

<p>CGC counting (7 mice, 3 sections/mouse) in left (PBS) and right eye (BAK).</p

Generation of a Muc5b-GFP KI mouse.

<p><b>(a)</b> Southern blot analysis of six (M1 to 6) heterozygous Flpe-excised KI mice. The genomic DNA of tested mice was compared to the DNA of a wild-type (WT) mouse. <i>Nhe</i>I-digested DNA was blotted onto a nylon membrane and hybridized with the external 3’ probe showing a 14.5-kb band for the WT allele and a 9.9-kb band for the Neo-excised allele (KI Muc5b-GFP) in agreement with the restriction map (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174764#pone.0174764.g001" target="_blank">Fig 1a</a>). <b>(b)</b> Example of tail-DNA genotyping by PCR.</p

Metastases following subcutaneous xenograft.

a<p>mice were injected with 10×10⁶ cells.</p>b<p>the presence of macrometastases was evaluated by histological analysis.</p>*<p><i>P</i> = 0.02. Mice injected with MCF7 clones/population expressing Mini5B were grouped and mice injected with clones/cell population not expressing Mini5B were grouped.</p

Histological analysis of mice sacrificed during the experimentation.

<p>(<b>A</b>) HE staining was performed on paraffin-embedded sections of the liver, lungs, and lymph nodes of mice injected with Mini5B–Luc clones. Metastasis is showed in the magnified image. (<b>B</b>) Immunofluorescence analysis was performed on paraffin-embedded sections of liver from mice injected with Mini5B–Luc clones and stained with anti-PCNA antibody. Metastatic cells were PCNA positive. (<b>C</b>) Double immunofluorescence analysis using the anti-human E-cadherin and anti-MUC5B antibodies were performed on paraffin-embedded sections of thoracic lymph nodes from mice injected with Mini5B–Luc clones. Metastatic cells expressed both the epithelial marker E-cadherin and secreted MUC5B protein. Nuclei were counterstained with Hoechst 33258 or propidium iodide. Mt, metastasis. Scale bar 50 µm.</p

Strategy for creating Muc5b-GFP knock-in (KI) mice and fluorescence stereo- and endomicroscopy.

<p><b>(a)</b><i>Muc5b</i> gene structure. Human and mouse <i>Muc5b</i> are composed of 49 exons indicated by rectangles (black, coding sequence and white, 3’UTR) and introns by lines. The large 31^st exon carries the sequences encoding peptides enriched in Ser+Thr+Pro. The initiation (ATG) and STOP codons are indicated. <b>(b)</b> Strategy for replacing the unique STOP codon of the endogenous mouse <i>Muc5b</i> gene with an enhanced GFP sequence (in green). A Gly-Ser-Ile-Ala-Thr linker is placed between the last amino acid of Muc5b and a synthetic sequence coding for a monomeric enhanced GFP sequence. Exons are indicated by rectangles and few of them are numbered. The unique 3’UTR region in black belongs to the 49^th and last exon. The targeting vector contains 3.2-kb of upstream (exons 38–47) and 2.2-kb of downstream sequences from the mouse Muc5b locus. LoxP sites (red vertical lines), which played no role in these experiments, flank the two last exons of <i>Muc5b</i>. A <i>Neo</i> positive selectable marker for embryonic stem cell integration flanked by two FRT sites (purple vertical lines) was inserted downstream of the Muc5b locus. The Muc5b-GFP line was obtained by crossing mice with a Flpe recombinase-expressing mouse in order to delete the neomycin cassette used for selection in embryonic stem cells. <b>(c)</b> Representative examples of pictures in bright-field mode and under GFP excitation by stereomicroscopy of fresh excised conjunctivas from wild-type (WT) and transgenic (Muc5b^GFP/+) mice. <b>(d)</b> Extracted frames from representative movies from conjunctivas acquired by pCLE (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174764#pone.0174764.s001" target="_blank">S1 Movie</a>) from a WT (+/+) and a transgenic mouse (ki/+).</p

Generation of stable clones expressing or not expressing the mini-mucin Mini5B.

<p>(<b>A</b>) Schematic representation of the control Ires–Luc and Mini5B–Luc vectors. The insert encoding the Mini5B was made by a signal sequence, 11 TR of 29 aa, one R-end domain of 111 aa, and two CYS domains of 110 aa. (<b>B</b>) Using the luciferase activity assay, four stable clones expressing the Ires–Luc transgene (clones B2, C5, C7, and C10) and two stable clones expressing the Mini5B–Luc transgene (clones A3 and D6) were obtained. (<b>C</b>) <i>MUC5B</i> mRNA expression was studied by qRT–PCR (TaqMan) in MCF7 parental cell line (white box), in MCF7 Ires-Luc clone (grey box) and in Mini5B-Luc clone (black box). Expression levels were normalized to mRNA levels of 18S and shown as x-fold relative to the normalized expression of <i>MUC5B</i> gene in MCF7 parental cells. Relative amounts of target genes were calculated using the ΔΔCt method. Values are means ± standard deviation from four to six independent samples. (<b>D</b>) MUC5B immunofluorescence analysis of MCF7 parental cells, Ires–Luc and Mini5B–Luc clones. Mini5B was secreted at the cell surface of Mini5B–Luc clones. Nuclei were counterstained with propidium iodide. Scale bar 50 µm. (<b>E</b>) Expression of the Mini5B was evaluated by RT–PCR. A PCR product of 159 bp was detected only in the Mini5B–Luc clone. (<b>F</b>) Western blot analysis of lysates from Ires–Luc clones, Mini5B–Luc clones, and Mini5B–Luc CYS shRNA subpopulation. (<b>G</b>) Inhibition of Mini5B production detected by Western blotting was quantified. Mini5B production of the clone Mini5B–Luc was set at 100 and the production of Ires–Luc was set at 0.</p