Journal: Oncogene

Article Title: Suppression of Tumorigenicity-14 , encoding matriptase, is a critical suppressor of colitis and colitis-associated colon carcinogenesis

doi: 10.1038/onc.2011.545

Figure Lengend Snippet: Expression of ST14 , encoding matriptase, in 14 gene expression array studies of human colon adenomas and adenocarcinomas. Data are expressed as fold change relative to corresponding normal tissue. * P< 0.05, ** P <0.01, *** P <0.001. See for details and references.

Article Snippet: The sections were blocked for 1 h in 5% bovine serum albumin (Sigma-Aldrich), or 10% horse serum (for matriptase IHC) in PBS, and incubated overnight at 4 C with primary antibody: matriptase (Sheep, Polyclonal, R&D Systems, Minneapolis, MN), cytokeratins (Rabbit, Polyclonal, DakoCytomation, Carpinteria, CA), LYVE-1 (Goat, Polyclonal, R&D Systems), β-catenin (Rabbit, Monoclonal, Cell Signaling, Danvers, MA), laminin (Rabbit, Polyclonal, Sigma-Aldrich), BrdU (Rat, Monoclonal, Accurate Chemicals & Scientific, Westbury, NY), CD3 (Rabbit, Polyclonal, DakoCytomation), κ-light chain (Rabbit, Polyclonal, DakoCytomation), Ki67 (Rabbit, Polyclonal, Novocastra, Westbury, NY), myeloperoxidase (Rabbit, Polyclonal, DakoCytomation), and Sox9 (Rabbit, Polyclonal, Millipore, Temecula, CA).

Techniques: Expressing

Journal: Oncogene

Article Title: Suppression of Tumorigenicity-14 , encoding matriptase, is a critical suppressor of colitis and colitis-associated colon carcinogenesis

doi: 10.1038/onc.2011.545

Figure Lengend Snippet: ( a , a ′) Immunohistochemical staining of eight week old St14 + ( a ) and littermate St14 − ( a ′) colons for β-catenin shows a membrane-associated β-catenin localization in St14 + epithelial cells ( arrows in a ), as compared to cytoplasmic and nuclear localization in adenocarcinomas of St14 − colons (examples with arrows in a ′). ( b , b ′) Immunohistochemical staining for the basement membrane marker laminin in 15 week old St14 + ( b ) and littermate St14 − ( b ′) mice shows the normal appearance of the basement membrane (example with arrow in b ) in St14 + mice. Loss of matriptase expression leads to increased deposition of laminin (examples with stars in b ′) and loss of normal structure of the basement membrane. ( c , c ′) Masson Trichrome staining of the colon of six week old St14 + ( c ) and littermate St14 − ( c ′) mice shows connective tissue in the submucosa of a normal colon (example with arrow in c ) and fibrosis of both the mucosa and submucosa of St14 − colon (examples with stars in c ′). ( d ) High magnification shows the cytological appearance of adenocarcinomas of St14 − mice. Atypical mitosis is shown by arrows . Scale bar = 200 μm ( a , a ′, b , b ′, c , c ′) and 20 μm ( d ).

Article Snippet: The sections were blocked for 1 h in 5% bovine serum albumin (Sigma-Aldrich), or 10% horse serum (for matriptase IHC) in PBS, and incubated overnight at 4 C with primary antibody: matriptase (Sheep, Polyclonal, R&D Systems, Minneapolis, MN), cytokeratins (Rabbit, Polyclonal, DakoCytomation, Carpinteria, CA), LYVE-1 (Goat, Polyclonal, R&D Systems), β-catenin (Rabbit, Monoclonal, Cell Signaling, Danvers, MA), laminin (Rabbit, Polyclonal, Sigma-Aldrich), BrdU (Rat, Monoclonal, Accurate Chemicals & Scientific, Westbury, NY), CD3 (Rabbit, Polyclonal, DakoCytomation), κ-light chain (Rabbit, Polyclonal, DakoCytomation), Ki67 (Rabbit, Polyclonal, Novocastra, Westbury, NY), myeloperoxidase (Rabbit, Polyclonal, DakoCytomation), and Sox9 (Rabbit, Polyclonal, Millipore, Temecula, CA).

Techniques: Immunohistochemical staining, Staining, Membrane, Marker, Expressing

Journal: Oncogene

Article Title: Suppression of Tumorigenicity-14 , encoding matriptase, is a critical suppressor of colitis and colitis-associated colon carcinogenesis

doi: 10.1038/onc.2011.545

Figure Lengend Snippet: ( a , a ′) BrdU staining of eight week old St14 + ( a ) and littermate St14 − ( a ′) mice shows proliferation restricted to the bottom of the crypts of normal colons (examples with arrows in a ). In St14 − colon, proliferating cells are found both in the bottom (examples with arrows in a ′) and distal parts of crypts (examples with arrowheads in a ′). (b,b ′ ) Periodic Acid-Schiff (PAS) staining of mucopolysaccharides produced by differentiated goblet cells in the colon of eleven week old St14 + ( b ) and littermate St14 − ( b ′) mice. Red staining shows mucin in the normal colon ( arrows in b ). Absence of red staining in ( b ′) indicates cessation of mucin production in matriptase-ablated colon. ( c , c ′, d , d ′) Immunohistochemical staining for T-cells ( c , c ′) and B-cells ( d , d ′) in, respectively, seven and 15 week old St14 + ( c , d ) and littermate St14 − ( c ′, d ′) colons. Baseline levels of T-and B-cells in the lamina propria of St14 + colon (examples with arrows in d and c ) and abundance of T- and B-cells in both mucosa and submucosa of St14 − colons (examples with stars in c ′, d ′). Scale bar = 100 μm.

Article Snippet: The sections were blocked for 1 h in 5% bovine serum albumin (Sigma-Aldrich), or 10% horse serum (for matriptase IHC) in PBS, and incubated overnight at 4 C with primary antibody: matriptase (Sheep, Polyclonal, R&D Systems, Minneapolis, MN), cytokeratins (Rabbit, Polyclonal, DakoCytomation, Carpinteria, CA), LYVE-1 (Goat, Polyclonal, R&D Systems), β-catenin (Rabbit, Monoclonal, Cell Signaling, Danvers, MA), laminin (Rabbit, Polyclonal, Sigma-Aldrich), BrdU (Rat, Monoclonal, Accurate Chemicals & Scientific, Westbury, NY), CD3 (Rabbit, Polyclonal, DakoCytomation), κ-light chain (Rabbit, Polyclonal, DakoCytomation), Ki67 (Rabbit, Polyclonal, Novocastra, Westbury, NY), myeloperoxidase (Rabbit, Polyclonal, DakoCytomation), and Sox9 (Rabbit, Polyclonal, Millipore, Temecula, CA).

Techniques: BrdU Staining, Staining, Produced, Immunohistochemical staining

Journal: Oncogene

Article Title: Suppression of Tumorigenicity-14 , encoding matriptase, is a critical suppressor of colitis and colitis-associated colon carcinogenesis

doi: 10.1038/onc.2011.545

Figure Lengend Snippet: The lumen of the colon and small intestine of weaning age St14 + and littermate St14 − animals was injected with Sulfo-NHS-LC-Biotin in PBS ( a , b , d , e ) or PBS ( c , f ). After three min, the intestine was excised, sectioned, and stained for biotin ( green ). Nuclei were stained with 4,6-diamino-2-phenylindol ( blue ). Arrows in a , d , and e show biotin bound to the surface of the mucosa. Arrowheads in b and the inset in e show biotin labeling of the basolateral membrane of polarized epithelial cells. The diffusion of biotin into intercellular space was not observed in the normal colon or small intestine ( a , d , also compare insets in d and e ). Stars show biotin labeling of connective tissue of both matriptase-ablated colon ( b ) and small intestine ( e ). There was no signal for biotin in colon and small intestine ( c , f ) injected with PBS. Scale bar = 20 μm.

Article Snippet: The sections were blocked for 1 h in 5% bovine serum albumin (Sigma-Aldrich), or 10% horse serum (for matriptase IHC) in PBS, and incubated overnight at 4 C with primary antibody: matriptase (Sheep, Polyclonal, R&D Systems, Minneapolis, MN), cytokeratins (Rabbit, Polyclonal, DakoCytomation, Carpinteria, CA), LYVE-1 (Goat, Polyclonal, R&D Systems), β-catenin (Rabbit, Monoclonal, Cell Signaling, Danvers, MA), laminin (Rabbit, Polyclonal, Sigma-Aldrich), BrdU (Rat, Monoclonal, Accurate Chemicals & Scientific, Westbury, NY), CD3 (Rabbit, Polyclonal, DakoCytomation), κ-light chain (Rabbit, Polyclonal, DakoCytomation), Ki67 (Rabbit, Polyclonal, Novocastra, Westbury, NY), myeloperoxidase (Rabbit, Polyclonal, DakoCytomation), and Sox9 (Rabbit, Polyclonal, Millipore, Temecula, CA).

Techniques: Injection, Staining, Labeling, Membrane, Diffusion-based Assay

Journal: Oncogene

Article Title: Suppression of Tumorigenicity-14 , encoding matriptase, is a critical suppressor of colitis and colitis-associated colon carcinogenesis

doi: 10.1038/onc.2011.545

Figure Lengend Snippet: Histological appearance of St14 + ( a – e ) and littermate St14 − ( a ′- e ′) colons at postnatal day 0 ( a , a ′), 5 ( b , b ′), 10 ( c , c ′), 15 ( d , d ′), and 20 ( e , e ′). No histological differences can be observed between normal and matriptase-ablated colon at days 0 and 5 (compare a and a ′, b and b ′). At day 10, St14 − colons show sporadic foci of detaching and apoptotic cells ( arrowheads in c ′). This phenotype is significantly stronger at days 15 and 20 with extensive anoikis ( arrowheads in d ′), apoptotic cells ( arrows in d ′, e ′), ulcerations ( arrowhead in e ′) and inflammatory cell infiltrates ( star in e ′). Scale bar = 100 μm.

Article Snippet: The sections were blocked for 1 h in 5% bovine serum albumin (Sigma-Aldrich), or 10% horse serum (for matriptase IHC) in PBS, and incubated overnight at 4 C with primary antibody: matriptase (Sheep, Polyclonal, R&D Systems, Minneapolis, MN), cytokeratins (Rabbit, Polyclonal, DakoCytomation, Carpinteria, CA), LYVE-1 (Goat, Polyclonal, R&D Systems), β-catenin (Rabbit, Monoclonal, Cell Signaling, Danvers, MA), laminin (Rabbit, Polyclonal, Sigma-Aldrich), BrdU (Rat, Monoclonal, Accurate Chemicals & Scientific, Westbury, NY), CD3 (Rabbit, Polyclonal, DakoCytomation), κ-light chain (Rabbit, Polyclonal, DakoCytomation), Ki67 (Rabbit, Polyclonal, Novocastra, Westbury, NY), myeloperoxidase (Rabbit, Polyclonal, DakoCytomation), and Sox9 (Rabbit, Polyclonal, Millipore, Temecula, CA).

Techniques:

Journal: Oncogene

Article Title: Suppression of Tumorigenicity-14 , encoding matriptase, is a critical suppressor of colitis and colitis-associated colon carcinogenesis

doi: 10.1038/onc.2011.545

Figure Lengend Snippet: Loss of matriptase from intestinal epithelium compromises epithelial barrier function thereby causing exposure of the commensal microbiota to resident immune cells. This triggers a repair response that includes activation of local inflammatory circuits and colonic stem cell activation. This response is perpetual, rather than transient, due to the intrinsic inability of matriptase-ablated to form a functional barrier. Persistent hyperproliferation of colonic stem cells within a DNA damaging chronic inflammatory microenvironment causes the formation of adenocarcinoma.

Article Snippet: The sections were blocked for 1 h in 5% bovine serum albumin (Sigma-Aldrich), or 10% horse serum (for matriptase IHC) in PBS, and incubated overnight at 4 C with primary antibody: matriptase (Sheep, Polyclonal, R&D Systems, Minneapolis, MN), cytokeratins (Rabbit, Polyclonal, DakoCytomation, Carpinteria, CA), LYVE-1 (Goat, Polyclonal, R&D Systems), β-catenin (Rabbit, Monoclonal, Cell Signaling, Danvers, MA), laminin (Rabbit, Polyclonal, Sigma-Aldrich), BrdU (Rat, Monoclonal, Accurate Chemicals & Scientific, Westbury, NY), CD3 (Rabbit, Polyclonal, DakoCytomation), κ-light chain (Rabbit, Polyclonal, DakoCytomation), Ki67 (Rabbit, Polyclonal, Novocastra, Westbury, NY), myeloperoxidase (Rabbit, Polyclonal, DakoCytomation), and Sox9 (Rabbit, Polyclonal, Millipore, Temecula, CA).

Techniques: Activation Assay, Functional Assay

Journal: Inflammatory Bowel Diseases

Article Title: Matriptase protects against experimental colitis and promotes intestinal barrier recovery

doi: 10.1002/ibd.21930

Figure Lengend Snippet: Matriptase is diminished in colonic epithelium of IBD patients. (A) qPCR analysis of human matriptase mRNA expression relative to GAPDH in 21 CD and 21 UC tissues collected from different individuals, compared with 6 normal tissues. Bars represent the average of mRNA expression normalized to GAPDH expression. (B) Immunostaining for human matriptase protein expression in normal human colon (a) and colonic tissues from CD (b,c) and UC patients (d-f). Arrow indicates area of patchy positive staining. Scale Bars, 100μM

Article Snippet: Antibodies were rabbit anti-human matriptase (Calbiochem), sheep anti-mouse matriptase and goat anti-HAI-1 (R&D Systems), rabbit anti-claudin-4 and rabbit anti-human claudin-2 (Zymed), rabbit anti-mouse claudin-2 (American Research Products), rabbit anti-GAPDH (Cell Signaling Technologies).

Techniques: Expressing, Immunostaining, Staining

Journal: Inflammatory Bowel Diseases

Article Title: Matriptase protects against experimental colitis and promotes intestinal barrier recovery

doi: 10.1002/ibd.21930

Figure Lengend Snippet: Matriptase expression changes in recovering intestinal epithelium. (A) qPCR analysis of St14 mRNA expression along the normal mouse GI tract. (B) qPCR analysis of St14 expression in segments of the jejunum and mid-distal colons of control mice during DSS-induced colitis. RNA signals are normalized to β-actin and expressed relative to the distal colon. n=3 mice per group, *p <0.05, **p <0.005. (C) Immunoblot of the jejunum and mid-distal colons of control mice (as in (B)) during DSS-induced colitis. Shown is the 70kDa full length matriptase.

Article Snippet: Antibodies were rabbit anti-human matriptase (Calbiochem), sheep anti-mouse matriptase and goat anti-HAI-1 (R&D Systems), rabbit anti-claudin-4 and rabbit anti-human claudin-2 (Zymed), rabbit anti-mouse claudin-2 (American Research Products), rabbit anti-GAPDH (Cell Signaling Technologies).

Techniques: Expressing, Control, Western Blot

Journal: Inflammatory Bowel Diseases

Article Title: Matriptase protects against experimental colitis and promotes intestinal barrier recovery

doi: 10.1002/ibd.21930

Figure Lengend Snippet: DSS induces decreased matriptase in colonic T84 epithelial monolayers. Polarized T84 monolayers (start TEER ~900 Ohms.cm2) were treated with or without 5% DSS in serum-free DME for 2.5 hrs, the DSS was removed, and the monolayers cultured in serum containing media for an additional 24 hrs. (A) (Upper panel) Changes in TEER. TEER was completely abolished after 2.5 hrs of DSS treatment. Upon removal of DSS (arrow), TEER values increased and approached that of untreated cultures by 24 hrs. (Lower panel) Immunoblot analysis showing that matriptase protein expression is significantly reduced (over 90%) upon exposure to DSS, and recovers upon removal of the DSS. (B) Matriptase siRNA knockdown in T84 monolayers compromises tight junction function. (Upper panel) Immunoblot analysis showing 80% knockdown of matriptase protein expression by 3 days. (Lower panel) TEER development is impaired in matriptase silenced T84 monolayers compared with control cells (siCtl vs siM2).

Article Snippet: Antibodies were rabbit anti-human matriptase (Calbiochem), sheep anti-mouse matriptase and goat anti-HAI-1 (R&D Systems), rabbit anti-claudin-4 and rabbit anti-human claudin-2 (Zymed), rabbit anti-mouse claudin-2 (American Research Products), rabbit anti-GAPDH (Cell Signaling Technologies).

Techniques: Cell Culture, Western Blot, Expressing, Knockdown, Control

Journal: Inflammatory Bowel Diseases

Article Title: Matriptase protects against experimental colitis and promotes intestinal barrier recovery

doi: 10.1002/ibd.21930

Figure Lengend Snippet: Loss of matriptase contributes to intestinal epithelial barrier disruption by inflammatory cytokines. (A) Polarized T84 monolayers on transwell filters were treated daily on the basolateral side with 10ng/ml TNFα, IL-4, or IL-13, 100U/ml IFNγ, a combination of TNFα and IFNγ, 10% macrophage (Mϕ) conditioned media or left untreated. Protein lysates were analyzed at 48hrs by immunoblotting for matriptase, HAI-1, claudin-2, claudin-4, and GAPDH. (B) Polarized T84 or (C) Caco-2 monolayers were treated for 48hrs with the indicated cytokines as in (A), then replaced with 5nM recombinant matriptase or media alone control. TEER was measured after 8 hrs.

Article Snippet: Antibodies were rabbit anti-human matriptase (Calbiochem), sheep anti-mouse matriptase and goat anti-HAI-1 (R&D Systems), rabbit anti-claudin-4 and rabbit anti-human claudin-2 (Zymed), rabbit anti-mouse claudin-2 (American Research Products), rabbit anti-GAPDH (Cell Signaling Technologies).

Techniques: Disruption, Western Blot, Recombinant, Control

Journal: PLOS Genetics

Article Title: Matriptase-dependent epidermal pre-neoplasm in zebrafish embryos caused by a combination of hypotonic stress and epithelial polarity defects

doi: 10.1371/journal.pgen.1010873

Figure Lengend Snippet: (A-C) Immunofluorescence on cross-sections of 54 hpf atp1b1a sibling (A), atp1b1a mutant (B), and atp1b1a mutant injected with st14a MO (C) showing pAKT (red) in basal cells in atp1b1a-/- but not in atp1b1a-/- , st14a MO (N = 3, n = 41–46). (D-F) Immunofluorescence on whole mounts, lateral views on trunk region for pRPS6 (red) and tp63 (green) in 54 hpf atp1b1a sibling (D), atp1b1a mutant (E), and atp1b1a mutant injected with st14a MO (F) (N = 3, n = 27–32). (G-I) Confocal images of the tail region of live 54 hpf embryos transgenic for NFkB-RE:eGFP injected with control MO (G), injected with atp1b1a MO (H) or co-injected with atp1b1a and st14a MO (I). Intensity of the GFP signal is color-coded. (J-L) Immunofluorescence of BrdU incorporation (red) of 56 hpf wild-type sibling (J), atp1b1a mutant (K), and atp1b1a mutant injected with st14a MO (L). (M-O) Whole mount in situ hybridization of mmp9 in 58 hpf wild-type sibling (M), atp1b1a mutant (N), or atp1b1a mutant injected with st14a MO (O) (N = 2, n = 18–24). (P) Quantification of GFP signal obtained from the fin fold region of embryos as shown in (G-I) (n = 3–5). (Q) Quantification of BrdU-positive cells in the fin fold area (n = 3–5). (R) RT-qPCR showing relative quantities of mmp9 transcript of 58 hpf atp1b1a-/- mutants in comparison to their atp1b1a+/+ and atp1b1a+/- siblings, either containing (bars 1+2) or lacking ( st14a-/- ; bars 3+4) functional Matriptase-1 (cDNA obtained from 15 pooled embryos each, N = 3). Significances in (P-R) were determined via a one-way ANOVA and Tukey’s post hoc test; ns, not significantly different (p>0.05); *,**,***,**** significantly different with p<0.05, p<0.01, p<0.001, p<0.0001, respectively. Scale bars: 10 μm (A,D,G,J,M).

Article Snippet: The following antibodies were used: mouse anti-GAPDH (Millipore, MAB374, 1:10000), goat anti-Hai1 (R&D Systems, AF1048-SP, 1 ug/ml), rabbit anti-Matriptase/MT-SP1 (Sigma Aldrich, IM1014-50UG, 1:1000), mouse anti-Scribble ([ ], clone 7C6.D10, 1:100), sheep anti-mouse HRP (Amersham, NA931V, 1:4000), donkey anti-rabbit HRP (Amersham, NA9340V, 1:4000), and rabbit anti-goat HRP (Invitrogen, 81–1620, 1:4000).

Techniques: Immunofluorescence, Mutagenesis, Injection, Transgenic Assay, BrdU Incorporation Assay, In Situ Hybridization, Quantitative RT-PCR, Functional Assay

Journal: PLOS Genetics

Article Title: Matriptase-dependent epidermal pre-neoplasm in zebrafish embryos caused by a combination of hypotonic stress and epithelial polarity defects

doi: 10.1371/journal.pgen.1010873

Figure Lengend Snippet: (A-B) RT-qPCR showing no significant change of st14a and hai1a transcript levels in atp1b1a mutants compared to their siblings (n = 3, cDNA obtained from pools of 15 embryos each at 56 hpf, significances were determined via Student’s t-test; ns, non-significant difference; p>0.05). (C-E) Reporter assay for Matriptase activity towards Par2 cleavage showing that zebrafish Matriptase1a cleaves AP-Par2b more efficiently at lower osmolalities. HEK293 cells were transfected with empty pcDNA3, pcDNA3+AP-Par2b, and pcDNA3+St14a. After 24 hrs in regular / isotonic medium (tonicity of 320 mOsm), cells were exposed to media of 320 mOsm, 270 mOsm, 230 mOsm, and 150 mOsm for 15 and 45 minutes, respectively. (C) At 15 min, absolute luminescence values of AP released into the supernatant progressively increase with increasing hypotonicity / lower tonicity. (D,E) Ratios of luminescence between isotonic and hypotonic media indicate an up to 3.79-fold increase (in 150 mOsm medium) after 15 min (D) and a 2.51-fold increase after 45 min (E). Ratios were determined from values as shown in (C), deducting baseline luminescence (bar 1 in C) from the luminenscences of co-transfected samples obtained at different osmolarities (bars 3–6 in C), normalized against the value at 320 mOsm (isotonic); n = 5, significances were determined via a one-way ANOVA and Tukey’s post hoc test; columns with same superscript letter (a,b,c) are not significantly different (p>0.05). (F) Immunoblot analysis for processed / activated Matriptase-1 (ST14) showing that culturing of MCF-10A cells for 24 hours in hypotonic medium (150 mOsm) leads to a 3.78-fold increase in active endogenous Matriptase-1 compared to cells cultured in isotonic medium (320 Osm). Bar diagram displays mean value of proteins normalized to loading control GAPDH (n = 3); ns, not significantly different (p>0.05); **, ***, significantly different with p<0.01, p<0.001, respectively. (G-H) Scribble knockout (SCRIB KO) in human MCF-10A cells does not affect protein levels of full length ST14 or HAI1. Representative western blots show unaltered amounts of endogenous full-length ST14 (G) and HAI1 (H) proteins in SCRIB-KO cells compared to knockout cells with re-introduced Scribble (SCRIB KO + SCRIB), cultured in media of 320 mOsm, 230 mOsm, or 150 mOsm for 1 hr. Bar diagrams display mean value of proteins normalized to loading control GAPDH (n = 2); all differences are not statistically significant (p>0.05). (I) Loss of Scribble increases active ST14 in media of different osmolalities. Representative western blots showing active ST14 and GAPDH of SCRIB-KO and SCRIB-KO + SCRIB cells cultured in media of 320 mOsm, 230 mOsm, and 150 mOsm for 24 hrs, with highest numbers in cells lacking the epithelial polarity protein Scribble protein and exposed to hypotonic stress. Bar diagram displays mean value of proteins normalized to loading control GAPDH (n = 3). Significances were determined via a one-way ANOVA and Tukey’s post hoc test; columns with same superscript letter (a,b,c,d) are not significantly different (p>0.05).

Article Snippet: The following antibodies were used: mouse anti-GAPDH (Millipore, MAB374, 1:10000), goat anti-Hai1 (R&D Systems, AF1048-SP, 1 ug/ml), rabbit anti-Matriptase/MT-SP1 (Sigma Aldrich, IM1014-50UG, 1:1000), mouse anti-Scribble ([ ], clone 7C6.D10, 1:100), sheep anti-mouse HRP (Amersham, NA931V, 1:4000), donkey anti-rabbit HRP (Amersham, NA9340V, 1:4000), and rabbit anti-goat HRP (Invitrogen, 81–1620, 1:4000).

Techniques: Quantitative RT-PCR, Reporter Assay, Activity Assay, Transfection, Western Blot, Cell Culture, Knock-Out

Journal: PLOS Genetics

Article Title: Matriptase-dependent epidermal pre-neoplasm in zebrafish embryos caused by a combination of hypotonic stress and epithelial polarity defects

doi: 10.1371/journal.pgen.1010873

Figure Lengend Snippet: (A-C) Loss of st14a in basal cells is neither necessary nor sufficient to normalize mmp9 expression in basal cells of atp1b1a morphants. (A) Schematic of experimental set up in which ventral ectodermal cells either from atp1b1a , st14a double morphant or atp1b1a morphant donors were homotopically transplanted at 6 hpf into the same region of atp1b1a or atp1b1a , st14a double morphant hosts, respectively ( atp1b1a MO, st14a MO> atp1b1a MO or atp1b1a MO> atp1b1a MO, st14a MO). Donor cells express Tg(Ola . Actb : Hsa . hras-egfp)- encoded membrane-tagged eGFP. Whole mount in situ hybridization for mmp9 (blue) and immunohistochemistry for eGFP (brown) of chimeric embryos at 58 hpf, showing that in atp1b1a MO, st14a MO> atp1b1a MO experiments, atp1b1a MO, st14a MO basal cells express mmp9 (B), and are thus not rescued although they lack functional Matriptase-1; whereas in atp1b1a MO> atp1b1a MO, st14a MO experiments, atp1b1a MO basal cells lack mmp9 expression (C), and are thus rescued although they contain Matriptase-1 (n = 6–8). (D-N) Re-introduction of st14a into peridermal cells of atp1b1a , st14a double morphants abrogates the rescue of basal cells, reflected by re-gained epidermal aggregate formation and enhanced mmp9 expression. (D) Immunofluorescence for eGFP-Matriptase1a (green), p63 as a nuclear marker for basal cells (red) and ZO1/Tjp1 as a marker for tight junctions in peridermal cells (red) on transverse section through the epidermis of a 48 hpf embryo transgenic for peri : Gal4 zc1044a and UAS : gfp-st14a fr58Tg , counterstained with DAPI (blue). Transgene-encoded Matriptase-1 is restricted to peridermal cells and localized at their basolateral membranes. (E-H’) Brightfield images of representative live 56 hpf embryos transgenic for peri : Gal4 zc1044a or peri : Gal4 zc1044a ; UAS : eGFP-st14a controls (E,F) and injected with atp1b1a and st14a morpholinos ( atp1b1a MO, st14a MO) (G,H); lateral views of entire embryos (E-H), and magnified views of tail region of same embryos (E’-H’). In contrast to atp1b1a MO, st14a MO embryos without transgenic re-introduction of st14a (G,G’), the atp1b1a MO, st14a MO embryos transgenic for peri : Gal4 zc1044a ; UAS : eGFP-st14a displays epidermal aggregates (H,H’), comparable to global atp1b1a single mutants (compare with L’). (I-L’) Representative whole mount mmp9 in situ hybridizations, revealing re-gained strong mmp9 expression in basal cells of 72 hpf atp1b1a MO, st14a MO embryo transgenic for peri : Gal4 zc1044a ; UAS : eGFP-st14a (L,L’, the latter counterstained for the basal cell marker p63), but not in the atp1b1a MO, st14a MO embryos lacking transgene-driven peridermal st14a re-expression (K). (M) Quantification of epidermal phenotype of 56 hpf embryos with respective genotypes, as shown in (E-H) (n = 20–46). (N) Quantification of mmp9 in situ signal of 72 hpf embryos with respective genotypes, as shown in (I-L) (n = 12–38). Scale bars: 20 μm (B,L’), 10 μm (D), 500 μm (E), 100 μm (E‘,I).

Article Snippet: The following antibodies were used: mouse anti-GAPDH (Millipore, MAB374, 1:10000), goat anti-Hai1 (R&D Systems, AF1048-SP, 1 ug/ml), rabbit anti-Matriptase/MT-SP1 (Sigma Aldrich, IM1014-50UG, 1:1000), mouse anti-Scribble ([ ], clone 7C6.D10, 1:100), sheep anti-mouse HRP (Amersham, NA931V, 1:4000), donkey anti-rabbit HRP (Amersham, NA9340V, 1:4000), and rabbit anti-goat HRP (Invitrogen, 81–1620, 1:4000).

Techniques: Expressing, In Situ Hybridization, Immunohistochemistry, Functional Assay, Immunofluorescence, Marker, Transgenic Assay, Injection, In Situ

Journal: PLOS Genetics

Article Title: Matriptase-dependent epidermal pre-neoplasm in zebrafish embryos caused by a combination of hypotonic stress and epithelial polarity defects

doi: 10.1371/journal.pgen.1010873

Figure Lengend Snippet: (A) Model of Matriptase activity restriction in the wild-type zebrafish epidermis. Hai1a is tightly associated with Matriptase, thereby inhibiting its activity, both in peridermal and basal cells. In addition, Matriptase trans-layer signaling from the periderm to the basal layer is restrained by confined levels of Matriptase at the basal side of peridermal cells. (B) Upon loss of Hai1a, Matriptase 1 is no longer inhibited (red star), leading to the cleavage of adjacent Par2b (orange asterisk), which in turn activates the EGFR-PLD pathway, to induce hyperproliferation and expression of mmp9 (a marker for EMT). Mild hypotonicity (small blue star), most likely due to compromised epidermal integrity, further enhances Matriptase activity levels. Note that additional pathways downstream of Par2b have been described, which lead to additional pre-neoplastic events, like sterile inflammation; however, they do not include PI3K . (C) More extreme hypotonicity in the pericellular space (due to loss of ATP1b1a or Pax2a; large blue star) causes (moderate) Matriptase activation even in the presence of Hai1a. This, however, only has subtle effects on epidermal cells ( , ), unless occurring in conjunction with the loss of epithelial polarity (due to loss of ATP1b1a or Lgl2), allowing Matriptase to shift towards the basal side of peridermal cells, thereby getting into physical contact and to cleave / activate Par2b and other, not yet identified targets (indicated by?) on underlying basal keratinocytes in trans. These targets activate a PI3K-pAKT-mTORC1-NFkB pathway in basal cells resulting in pre-neoplastic events like hyperproliferation, EMT and, in contrast to hai1a mutants, strong invasiveness of basal cells.

Article Snippet: The following antibodies were used: mouse anti-GAPDH (Millipore, MAB374, 1:10000), goat anti-Hai1 (R&D Systems, AF1048-SP, 1 ug/ml), rabbit anti-Matriptase/MT-SP1 (Sigma Aldrich, IM1014-50UG, 1:1000), mouse anti-Scribble ([ ], clone 7C6.D10, 1:100), sheep anti-mouse HRP (Amersham, NA931V, 1:4000), donkey anti-rabbit HRP (Amersham, NA9340V, 1:4000), and rabbit anti-goat HRP (Invitrogen, 81–1620, 1:4000).

Techniques: Activity Assay, Expressing, Marker, Activation Assay