mt2 antibodies Search Results


mt2  (Alomone Labs)
93
Alomone Labs mt2
Mt2, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 93 stars, based on 1 article reviews
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mt2 mmp  (R&D Systems)
93
R&D Systems mt2 mmp
<t>MT2-MMP</t> associates with ZO-1 in polarized MDCK cells. (A) Western blot analysis of HA, E-cadherin and Rho-GDI in biotinylated cell lysates from Mock, MT2-MMP (MT2FL) and MT2-MMPWK (MT2WK) stable MDCK transfectants pulled down with streptavidin beads; input, unbound and bound fractions are shown (Inp, Unb, Biot). (B) Western blot analysis of ZO-1 and HA in cell lysates from Mock, MT2-MMP and MT2-MMPWK stable MDCK transfectants pulled down with anti-HA antibody; IgG immunoprecipitates and whole lysates (Input) are also shown as controls. A blot of the input lanes after a longer exposure is also shown. (C) Representative maximal projections from apical and basolateral stacks of confocal sections from polarized MDCK transfectants stained for HA (MT2-MMP, green), ZO-1 (red) and nuclei (Hoechst, blue). (D) Orthogonal x–z views of 3D confocal image stacks from C. (E) Representative peak intensity profiles from x–z views of 3D confocal image stacks from C. Graph to the right shows the quantification of MT2-MMP/ZO-1 Pearson correlation coefficient in polarized MT2-FL and MT2-WK MDCK transfectants. Values are mean±s.e.m. n=40 cells per condition in two independent experiments; *P<0.05.
Mt2 Mmp, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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anti mt2a ab  (Proteintech)
90
Proteintech anti mt2a ab
Fig. 3. The cytotoxicity of Cd and intracellular Cd accumulation with or without GOs pre-treatment. (a) The cellular viability of BEAS-2B cells detected by CCK8 assay after being treated to Cd (1, 5, 10, 20, 25, 30 and 50 μM) for 24 h (n = 5, * indicate P < 0.05, compared to the untreated group). (b) The heat map of the cell viability inspected by CCK8 assay for BEAS- 2B cells after being treated to Cd (1, 5, 10, 20, 25, 30 and 50 μM) for 24 h with or without pre-treatment of P-GO, A-GO, or G-GO at the dose of 10 μg/mL, respectively (n = 5). (c) Intracellular Cd mass quan tification by ICP-MS. BEAS-2B cells were pre-treated with P-GO, A-GO, G-GO at 10 μg/mL for 24 h, and then exposed to CdCl2 at 10 μM for 24 h (n = 5), *P < 0.05. (d) Western blot analysis of MT1M and <t>MT2A</t> protein expression levels in BEAS-2B cells exposed to either GOs (10 μg/mL) or Cd (10 μM) or a combina tion of both (GOs + Cd) for 24 h.
Anti Mt2a Ab, supplied by Proteintech, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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human mt2 mmp  (R&D Systems)
94
R&D Systems human mt2 mmp
HEK293 cells transfected with <t>MT1-MMP</t> cDNA invade 3D collagen gels in response to LPA . (A) Tumor cell lysates were prepared for Western blot analysis. Lysates were probed for MT1-MMP to assess protein expression in the four tumor cell lines. Lysates were probed for Actin as a loading control. (B) HEK293 cells were transfected with the pAdTrack-CMV plasmid as a control, or plasmids encoding MT1-MMP, <t>MT2-MMP,</t> or MT3-MMP cDNA 24 hours prior to placement in invasion assays. Cells were allowed to invade 2.0 mg/ml collagen gels in the presence or absence of 1 μM LPA. Data are expressed as mean numbers of invading cells per HPF (20×) (± S.D.) from a minimum of 20 fields. (C) Lysates from HEK293 cells transfected with cDNAs encoding the designated genes were prepared for Western blot analysis and probed for GFP, MT1-MMP, MT2-MMP, MT3-MMP, or Actin as a loading control. TRK = pAdTrack-CMV, MT1 = MT1-MMP, MT2 = MT2-MMP, MT3 = MT3-MMP.
Human Mt2 Mmp, supplied by R&D Systems, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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rabbit anti rat mt2 antibody  (Biorbyt)
92
Biorbyt rabbit anti rat mt2 antibody
FIGURE 5 | Representative western blots showing (A) melatonin receptor-1 (MT1) (37 kDa), and −2 <t>(MT2)</t> (40 kDa), and RORα (59 kDa) bands in control, prenatal dexamethasone exposure (DEX), postnatal high-fat diet (HF), prenatal DEX plus postnatal (DEX+HF), and melatonin treatment (DEX+HF+M) offspring rats at 16 weeks of age. Relative abundance of renal cortical (B) MT1, (C) MT2, and (D) RORα as quantified. (E) Effect of DEX, HF, and melatonin on renal melatonin level. N = 6/group. *P < 0.05 vs. control; #P < 0.05 vs. DEX; ‡P < 0.05 vs. DEX+HF.
Rabbit Anti Rat Mt2 Antibody, supplied by Biorbyt, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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mt2 antibody  (ABclonal Biotechnology)
90
ABclonal Biotechnology mt2 antibody
XBP1 expression in macrophages is increased and positively correlates with IR-induced liver injury in patients. Liver tissues and macrophages were harvested from 25 patients undergoing PH. ( A-D ) Prehepatectomy/posthepatectomy <t>XBP1s</t> expression profiles in liver tissues and macrophages (representative 6 cases) through Western blotting assay. ( B, C ) Expressions of XBP1 mRNA in liver tissues and in liver macrophages. ( E ) Dual-immunofluorescence staining for XBP1 ( green ) and CD68 ( red ) colocalization in human liver tissues, which were also subjected to HE and TUNEL analysis. ( F ) Twenty-five patients were divided into the low (n = 12) and high (n = 13) groups using the postoperative median XBP1/GAPDH ratio as the threshold. ( G ) The ratio of posthepatectomy XBP1/GAPDH positively correlated with serum ALT and AST levels at POD1. ( H ) Serum ALT and AST levels were measured at POD1 in both low and high groups. ∗ P < .05 by Student t test.
Mt2 Antibody, supplied by ABclonal Biotechnology, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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antibodies against ifn-alpha  (Mabtech Inc)
90
Mabtech Inc antibodies against ifn-alpha
STAT phosphorylation <t>and</t> <t>IFN‐I</t> dependent NK cell activation following reovirus treatment. (a) STAT phosphorylation in CD56 bright and CD56 dim NK cells (detected by intracellular staining and flow cytometry) in PBMC cultured without virus (untreated; black line) or with 1 MOI reovirus (purple line), for 8, 24 and 48 h. Graphs show mean MFI and standard deviation from three donors. Data were analysed by two‐way repeated‐measures ANOVA, followed by Sidak multiple comparisons test. * p < 0·05 ** p < 0·01. (b) NK cell activation by reovirus is IFN‐I dependent. The flow chart shows the approach taken; PBMC (from one donor) were left untreated or treated with reovirus for 24 h and the conditioned media (CM) filtered to remove viruses. CM was added to purified NK cells in the presence of an IFN‐I blocking antibody cocktail (IFN block), a control blocking cocktail (control block) or no added antibody (no block). CM from untreated PBMC was used a control. After 48 h, the NK cell surface expression of CD69 and tetherin was measured by flow cytometry. Data is from control CM or CM from reovirus‐treated PBMC from a single donor, applied to three NK cell donors. The y ‐axes show the percentage of CD69 expressing cells (top panel) or the fold change in MFI of tetherin relative to control CM and no added antibody treatment (bottom panel), due to constitutive low‐level expression of this molecule on unstimulated NK cells . Differences between mean percentage positive values for CD69, or mean fold change MFI for tetherin, were analysed by two‐way repeated‐measures ANOVA, followed by Sidak multiple comparisons test. * p < 0·05 ** p < 0·01
Antibodies Against Ifn Alpha, supplied by Mabtech Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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anti-μ-opioid receptor antibody mt-2 #2148  (Valeant Pharmaceuticals)
90
Valeant Pharmaceuticals anti-μ-opioid receptor antibody mt-2 #2148
STAT phosphorylation <t>and</t> <t>IFN‐I</t> dependent NK cell activation following reovirus treatment. (a) STAT phosphorylation in CD56 bright and CD56 dim NK cells (detected by intracellular staining and flow cytometry) in PBMC cultured without virus (untreated; black line) or with 1 MOI reovirus (purple line), for 8, 24 and 48 h. Graphs show mean MFI and standard deviation from three donors. Data were analysed by two‐way repeated‐measures ANOVA, followed by Sidak multiple comparisons test. * p < 0·05 ** p < 0·01. (b) NK cell activation by reovirus is IFN‐I dependent. The flow chart shows the approach taken; PBMC (from one donor) were left untreated or treated with reovirus for 24 h and the conditioned media (CM) filtered to remove viruses. CM was added to purified NK cells in the presence of an IFN‐I blocking antibody cocktail (IFN block), a control blocking cocktail (control block) or no added antibody (no block). CM from untreated PBMC was used a control. After 48 h, the NK cell surface expression of CD69 and tetherin was measured by flow cytometry. Data is from control CM or CM from reovirus‐treated PBMC from a single donor, applied to three NK cell donors. The y ‐axes show the percentage of CD69 expressing cells (top panel) or the fold change in MFI of tetherin relative to control CM and no added antibody treatment (bottom panel), due to constitutive low‐level expression of this molecule on unstimulated NK cells . Differences between mean percentage positive values for CD69, or mean fold change MFI for tetherin, were analysed by two‐way repeated‐measures ANOVA, followed by Sidak multiple comparisons test. * p < 0·05 ** p < 0·01
Anti μ Opioid Receptor Antibody Mt 2 #2148, supplied by Valeant Pharmaceuticals, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/anti-μ-opioid receptor antibody mt-2 #2148/product/Valeant Pharmaceuticals
Average 90 stars, based on 1 article reviews
anti-μ-opioid receptor antibody mt-2 #2148 - by Bioz Stars, 2026-03
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mt1 mt2  (MBL Life science)
90
MBL Life science mt1 mt2
STAT phosphorylation <t>and</t> <t>IFN‐I</t> dependent NK cell activation following reovirus treatment. (a) STAT phosphorylation in CD56 bright and CD56 dim NK cells (detected by intracellular staining and flow cytometry) in PBMC cultured without virus (untreated; black line) or with 1 MOI reovirus (purple line), for 8, 24 and 48 h. Graphs show mean MFI and standard deviation from three donors. Data were analysed by two‐way repeated‐measures ANOVA, followed by Sidak multiple comparisons test. * p < 0·05 ** p < 0·01. (b) NK cell activation by reovirus is IFN‐I dependent. The flow chart shows the approach taken; PBMC (from one donor) were left untreated or treated with reovirus for 24 h and the conditioned media (CM) filtered to remove viruses. CM was added to purified NK cells in the presence of an IFN‐I blocking antibody cocktail (IFN block), a control blocking cocktail (control block) or no added antibody (no block). CM from untreated PBMC was used a control. After 48 h, the NK cell surface expression of CD69 and tetherin was measured by flow cytometry. Data is from control CM or CM from reovirus‐treated PBMC from a single donor, applied to three NK cell donors. The y ‐axes show the percentage of CD69 expressing cells (top panel) or the fold change in MFI of tetherin relative to control CM and no added antibody treatment (bottom panel), due to constitutive low‐level expression of this molecule on unstimulated NK cells . Differences between mean percentage positive values for CD69, or mean fold change MFI for tetherin, were analysed by two‐way repeated‐measures ANOVA, followed by Sidak multiple comparisons test. * p < 0·05 ** p < 0·01
Mt1 Mt2, supplied by MBL Life science, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/mt1 mt2/product/MBL Life science
Average 90 stars, based on 1 article reviews
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monoclonal antibodies against mt2-mmp (162–22g5)  (Oncogene Science Inc)
90
Oncogene Science Inc monoclonal antibodies against mt2-mmp (162–22g5)
STAT phosphorylation <t>and</t> <t>IFN‐I</t> dependent NK cell activation following reovirus treatment. (a) STAT phosphorylation in CD56 bright and CD56 dim NK cells (detected by intracellular staining and flow cytometry) in PBMC cultured without virus (untreated; black line) or with 1 MOI reovirus (purple line), for 8, 24 and 48 h. Graphs show mean MFI and standard deviation from three donors. Data were analysed by two‐way repeated‐measures ANOVA, followed by Sidak multiple comparisons test. * p < 0·05 ** p < 0·01. (b) NK cell activation by reovirus is IFN‐I dependent. The flow chart shows the approach taken; PBMC (from one donor) were left untreated or treated with reovirus for 24 h and the conditioned media (CM) filtered to remove viruses. CM was added to purified NK cells in the presence of an IFN‐I blocking antibody cocktail (IFN block), a control blocking cocktail (control block) or no added antibody (no block). CM from untreated PBMC was used a control. After 48 h, the NK cell surface expression of CD69 and tetherin was measured by flow cytometry. Data is from control CM or CM from reovirus‐treated PBMC from a single donor, applied to three NK cell donors. The y ‐axes show the percentage of CD69 expressing cells (top panel) or the fold change in MFI of tetherin relative to control CM and no added antibody treatment (bottom panel), due to constitutive low‐level expression of this molecule on unstimulated NK cells . Differences between mean percentage positive values for CD69, or mean fold change MFI for tetherin, were analysed by two‐way repeated‐measures ANOVA, followed by Sidak multiple comparisons test. * p < 0·05 ** p < 0·01
Monoclonal Antibodies Against Mt2 Mmp (162–22g5), supplied by Oncogene Science Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/monoclonal antibodies against mt2-mmp (162–22g5)/product/Oncogene Science Inc
Average 90 stars, based on 1 article reviews
monoclonal antibodies against mt2-mmp (162–22g5) - by Bioz Stars, 2026-03
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mtnr1b polyclonal antibody  (Bioss)
94
Bioss mtnr1b polyclonal antibody
STAT phosphorylation <t>and</t> <t>IFN‐I</t> dependent NK cell activation following reovirus treatment. (a) STAT phosphorylation in CD56 bright and CD56 dim NK cells (detected by intracellular staining and flow cytometry) in PBMC cultured without virus (untreated; black line) or with 1 MOI reovirus (purple line), for 8, 24 and 48 h. Graphs show mean MFI and standard deviation from three donors. Data were analysed by two‐way repeated‐measures ANOVA, followed by Sidak multiple comparisons test. * p < 0·05 ** p < 0·01. (b) NK cell activation by reovirus is IFN‐I dependent. The flow chart shows the approach taken; PBMC (from one donor) were left untreated or treated with reovirus for 24 h and the conditioned media (CM) filtered to remove viruses. CM was added to purified NK cells in the presence of an IFN‐I blocking antibody cocktail (IFN block), a control blocking cocktail (control block) or no added antibody (no block). CM from untreated PBMC was used a control. After 48 h, the NK cell surface expression of CD69 and tetherin was measured by flow cytometry. Data is from control CM or CM from reovirus‐treated PBMC from a single donor, applied to three NK cell donors. The y ‐axes show the percentage of CD69 expressing cells (top panel) or the fold change in MFI of tetherin relative to control CM and no added antibody treatment (bottom panel), due to constitutive low‐level expression of this molecule on unstimulated NK cells . Differences between mean percentage positive values for CD69, or mean fold change MFI for tetherin, were analysed by two‐way repeated‐measures ANOVA, followed by Sidak multiple comparisons test. * p < 0·05 ** p < 0·01
Mtnr1b Polyclonal Antibody, supplied by Bioss, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 94 stars, based on 1 article reviews
mtnr1b polyclonal antibody - by Bioz Stars, 2026-03
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mt2 monoclonal antibodies  (MBL Life science)
90
MBL Life science mt2 monoclonal antibodies
STAT phosphorylation <t>and</t> <t>IFN‐I</t> dependent NK cell activation following reovirus treatment. (a) STAT phosphorylation in CD56 bright and CD56 dim NK cells (detected by intracellular staining and flow cytometry) in PBMC cultured without virus (untreated; black line) or with 1 MOI reovirus (purple line), for 8, 24 and 48 h. Graphs show mean MFI and standard deviation from three donors. Data were analysed by two‐way repeated‐measures ANOVA, followed by Sidak multiple comparisons test. * p < 0·05 ** p < 0·01. (b) NK cell activation by reovirus is IFN‐I dependent. The flow chart shows the approach taken; PBMC (from one donor) were left untreated or treated with reovirus for 24 h and the conditioned media (CM) filtered to remove viruses. CM was added to purified NK cells in the presence of an IFN‐I blocking antibody cocktail (IFN block), a control blocking cocktail (control block) or no added antibody (no block). CM from untreated PBMC was used a control. After 48 h, the NK cell surface expression of CD69 and tetherin was measured by flow cytometry. Data is from control CM or CM from reovirus‐treated PBMC from a single donor, applied to three NK cell donors. The y ‐axes show the percentage of CD69 expressing cells (top panel) or the fold change in MFI of tetherin relative to control CM and no added antibody treatment (bottom panel), due to constitutive low‐level expression of this molecule on unstimulated NK cells . Differences between mean percentage positive values for CD69, or mean fold change MFI for tetherin, were analysed by two‐way repeated‐measures ANOVA, followed by Sidak multiple comparisons test. * p < 0·05 ** p < 0·01
Mt2 Monoclonal Antibodies, supplied by MBL Life science, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/mt2 monoclonal antibodies/product/MBL Life science
Average 90 stars, based on 1 article reviews
mt2 monoclonal antibodies - by Bioz Stars, 2026-03
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Image Search Results


MT2-MMP associates with ZO-1 in polarized MDCK cells. (A) Western blot analysis of HA, E-cadherin and Rho-GDI in biotinylated cell lysates from Mock, MT2-MMP (MT2FL) and MT2-MMPWK (MT2WK) stable MDCK transfectants pulled down with streptavidin beads; input, unbound and bound fractions are shown (Inp, Unb, Biot). (B) Western blot analysis of ZO-1 and HA in cell lysates from Mock, MT2-MMP and MT2-MMPWK stable MDCK transfectants pulled down with anti-HA antibody; IgG immunoprecipitates and whole lysates (Input) are also shown as controls. A blot of the input lanes after a longer exposure is also shown. (C) Representative maximal projections from apical and basolateral stacks of confocal sections from polarized MDCK transfectants stained for HA (MT2-MMP, green), ZO-1 (red) and nuclei (Hoechst, blue). (D) Orthogonal x–z views of 3D confocal image stacks from C. (E) Representative peak intensity profiles from x–z views of 3D confocal image stacks from C. Graph to the right shows the quantification of MT2-MMP/ZO-1 Pearson correlation coefficient in polarized MT2-FL and MT2-WK MDCK transfectants. Values are mean±s.e.m. n=40 cells per condition in two independent experiments; *P<0.05.

Journal: Journal of Cell Science

Article Title: E-cadherin cleavage by MT2-MMP regulates apical junctional signaling and epithelial homeostasis in the intestine

doi: 10.1242/jcs.203687

Figure Lengend Snippet: MT2-MMP associates with ZO-1 in polarized MDCK cells. (A) Western blot analysis of HA, E-cadherin and Rho-GDI in biotinylated cell lysates from Mock, MT2-MMP (MT2FL) and MT2-MMPWK (MT2WK) stable MDCK transfectants pulled down with streptavidin beads; input, unbound and bound fractions are shown (Inp, Unb, Biot). (B) Western blot analysis of ZO-1 and HA in cell lysates from Mock, MT2-MMP and MT2-MMPWK stable MDCK transfectants pulled down with anti-HA antibody; IgG immunoprecipitates and whole lysates (Input) are also shown as controls. A blot of the input lanes after a longer exposure is also shown. (C) Representative maximal projections from apical and basolateral stacks of confocal sections from polarized MDCK transfectants stained for HA (MT2-MMP, green), ZO-1 (red) and nuclei (Hoechst, blue). (D) Orthogonal x–z views of 3D confocal image stacks from C. (E) Representative peak intensity profiles from x–z views of 3D confocal image stacks from C. Graph to the right shows the quantification of MT2-MMP/ZO-1 Pearson correlation coefficient in polarized MT2-FL and MT2-WK MDCK transfectants. Values are mean±s.e.m. n=40 cells per condition in two independent experiments; *P<0.05.

Article Snippet: Antibodies Antibodies used were against β-actin (Sigma-Aldrich, A5441), GST (Thermo Fisher Scientific, A5800), HA (Covance, MMS-101P), MT1-MMP (LEM2/63; Gálvez et al., 2001 ), MT2-MMP (R&D Systems, MAB9161; and a rabbit polyclonal antibody generated by our group at CNB, Madrid, Spain, directed against 16 aa of hMT2 DEPWTFSSTDLHGNNL), Tubulin (Sigma, T6074), pSrc (Cell Signaling, 2101), ZO-1 (Thermo Fisher Scientific, 40-2300), E-cadherin (Cell Signaling, 3195 and BD Biosciences, 610181), Hoechst 3342 (Thermo Fisher Scientific), Ki67 (Abcam, ab16667), Phalloidin 647 (Thermo Fisher Scientific, {"type":"entrez-nucleotide","attrs":{"text":"A22287","term_id":"641467"}} A22287 ), Myosin IIB (Santa Cruz Biotechnology, sc-15370), β-catenin (BD Biosciences, 610153), Rho-GDI (Santa Cruz Biotechnology, sc-360), Ezrin (Upstate, 07-130), EEA1 (Santa Cruz Biotechnology, sc-6415), TfR (Invitrogen, H68.4), HGS (Abcam, ab72053), TSG101 (Abcam, ab30871).

Techniques: Western Blot, Staining

MT2-MMP overexpression induces aberrant apical epithelial cell accumulation in polarized MDCK monolayers. (A) Orthogonal x–z projections of 3D confocal image stacks of MDCK transfectants stained for F-actin (Phalloidin, gray), HA (MT2-MMP, green) and Hoechst (nuclei, blue). Scale bar: 10 μm. (B) Quantification of apical epithelial foci per field (left) and the percentage of foci having more than 8 nuclei (right). 10 fields were counted per condition in n=4 independent experiments. (C) Representative maximal projections are shown from subapical and complete stacks of confocal sections from polarized MDCK transfectants stained for E-cadherin (gray). The dotted yellow line marks apical foci. Orthogonal x–z views are shown to the right. (D) Line and bar graphs show E-cadherin peak and average mean fluorescence intensity (MFI), respectively, around the junctions formed by MDCK transfectants. Data are represented as mean±s.e.m. and were tested by one-way ANOVA versus mock 1 followed by Dunnett's post-test in B and C. **P<0.01, ***P<0.001, ****P<0.001; ns, not significant.

Journal: Journal of Cell Science

Article Title: E-cadherin cleavage by MT2-MMP regulates apical junctional signaling and epithelial homeostasis in the intestine

doi: 10.1242/jcs.203687

Figure Lengend Snippet: MT2-MMP overexpression induces aberrant apical epithelial cell accumulation in polarized MDCK monolayers. (A) Orthogonal x–z projections of 3D confocal image stacks of MDCK transfectants stained for F-actin (Phalloidin, gray), HA (MT2-MMP, green) and Hoechst (nuclei, blue). Scale bar: 10 μm. (B) Quantification of apical epithelial foci per field (left) and the percentage of foci having more than 8 nuclei (right). 10 fields were counted per condition in n=4 independent experiments. (C) Representative maximal projections are shown from subapical and complete stacks of confocal sections from polarized MDCK transfectants stained for E-cadherin (gray). The dotted yellow line marks apical foci. Orthogonal x–z views are shown to the right. (D) Line and bar graphs show E-cadherin peak and average mean fluorescence intensity (MFI), respectively, around the junctions formed by MDCK transfectants. Data are represented as mean±s.e.m. and were tested by one-way ANOVA versus mock 1 followed by Dunnett's post-test in B and C. **P<0.01, ***P<0.001, ****P<0.001; ns, not significant.

Article Snippet: Antibodies Antibodies used were against β-actin (Sigma-Aldrich, A5441), GST (Thermo Fisher Scientific, A5800), HA (Covance, MMS-101P), MT1-MMP (LEM2/63; Gálvez et al., 2001 ), MT2-MMP (R&D Systems, MAB9161; and a rabbit polyclonal antibody generated by our group at CNB, Madrid, Spain, directed against 16 aa of hMT2 DEPWTFSSTDLHGNNL), Tubulin (Sigma, T6074), pSrc (Cell Signaling, 2101), ZO-1 (Thermo Fisher Scientific, 40-2300), E-cadherin (Cell Signaling, 3195 and BD Biosciences, 610181), Hoechst 3342 (Thermo Fisher Scientific), Ki67 (Abcam, ab16667), Phalloidin 647 (Thermo Fisher Scientific, {"type":"entrez-nucleotide","attrs":{"text":"A22287","term_id":"641467"}} A22287 ), Myosin IIB (Santa Cruz Biotechnology, sc-15370), β-catenin (BD Biosciences, 610153), Rho-GDI (Santa Cruz Biotechnology, sc-360), Ezrin (Upstate, 07-130), EEA1 (Santa Cruz Biotechnology, sc-6415), TfR (Invitrogen, H68.4), HGS (Abcam, ab72053), TSG101 (Abcam, ab30871).

Techniques: Over Expression, Staining, Fluorescence

Apical epithelial cell accumulation depends on MT2-MMP catalytic activity. (A) Representative maximal projections from confocal sections of polarized MDCK transfectants stained for E-cadherin (gray) in the presence of GM6001 (50 μM) or vehicle (DMSO). Orthogonal x–z views are shown below. (B) Line and bar graphs show E-cadherin peak and average intensity, respectively, around the junctions formed by MDCK transfectants treated as in A. Bar graph at the bottom shows the number of apical events on the polarized MDCK monolayer in the presence or absence of DMSO. In the middle and bottom graphs, the difference between mock DMSO and MT2 FL were significant with P<0.01 and P<0.0001, respectively. (C) Representative maximal projections are shown from confocal sections of polarized MDCK transfectants (mock, MT2 and MT2EA) stained for E-cadherin (gray). Orthogonal x–z views are shown to the right. (D) Line and bar graphs show E-cadherin peak and average mean fluorescence intensity (MFI), respectively, around the junctions formed by MDCK transfectants shown in C. Bar graph on the right shows the number of apical events occurring in polarized MDCK monolayers. Data are represented as mean± s.e.m. and were tested by one-way ANOVA followed by Sidak post-test in B. Dunnett's post-test was used in D. *P<0.05, **P<0.01, ****P<0.001; ns, not significant.

Journal: Journal of Cell Science

Article Title: E-cadherin cleavage by MT2-MMP regulates apical junctional signaling and epithelial homeostasis in the intestine

doi: 10.1242/jcs.203687

Figure Lengend Snippet: Apical epithelial cell accumulation depends on MT2-MMP catalytic activity. (A) Representative maximal projections from confocal sections of polarized MDCK transfectants stained for E-cadherin (gray) in the presence of GM6001 (50 μM) or vehicle (DMSO). Orthogonal x–z views are shown below. (B) Line and bar graphs show E-cadherin peak and average intensity, respectively, around the junctions formed by MDCK transfectants treated as in A. Bar graph at the bottom shows the number of apical events on the polarized MDCK monolayer in the presence or absence of DMSO. In the middle and bottom graphs, the difference between mock DMSO and MT2 FL were significant with P<0.01 and P<0.0001, respectively. (C) Representative maximal projections are shown from confocal sections of polarized MDCK transfectants (mock, MT2 and MT2EA) stained for E-cadherin (gray). Orthogonal x–z views are shown to the right. (D) Line and bar graphs show E-cadherin peak and average mean fluorescence intensity (MFI), respectively, around the junctions formed by MDCK transfectants shown in C. Bar graph on the right shows the number of apical events occurring in polarized MDCK monolayers. Data are represented as mean± s.e.m. and were tested by one-way ANOVA followed by Sidak post-test in B. Dunnett's post-test was used in D. *P<0.05, **P<0.01, ****P<0.001; ns, not significant.

Article Snippet: Antibodies Antibodies used were against β-actin (Sigma-Aldrich, A5441), GST (Thermo Fisher Scientific, A5800), HA (Covance, MMS-101P), MT1-MMP (LEM2/63; Gálvez et al., 2001 ), MT2-MMP (R&D Systems, MAB9161; and a rabbit polyclonal antibody generated by our group at CNB, Madrid, Spain, directed against 16 aa of hMT2 DEPWTFSSTDLHGNNL), Tubulin (Sigma, T6074), pSrc (Cell Signaling, 2101), ZO-1 (Thermo Fisher Scientific, 40-2300), E-cadherin (Cell Signaling, 3195 and BD Biosciences, 610181), Hoechst 3342 (Thermo Fisher Scientific), Ki67 (Abcam, ab16667), Phalloidin 647 (Thermo Fisher Scientific, {"type":"entrez-nucleotide","attrs":{"text":"A22287","term_id":"641467"}} A22287 ), Myosin IIB (Santa Cruz Biotechnology, sc-15370), β-catenin (BD Biosciences, 610153), Rho-GDI (Santa Cruz Biotechnology, sc-360), Ezrin (Upstate, 07-130), EEA1 (Santa Cruz Biotechnology, sc-6415), TfR (Invitrogen, H68.4), HGS (Abcam, ab72053), TSG101 (Abcam, ab30871).

Techniques: Activity Assay, Staining, Fluorescence

E-cadherin is cleaved by MT2-MMP after N445 in the EC5 loop. (A) In silico model of canine E-cadherin (green)/human MT2-MMP (blue) interactions in cis association at the plasma membrane; the catalytic MT2-MMP center and the E-cadherin peptide, GPIPEPRNMDFCQKNPQP, are shown in orange and red, respectively. (B) Scheme of E-cadherin structure with the peptide containing the predicted cleavage sites after N445 and N459 in the EC5 loop. (C) Representative extracted ion chromatograms of 3 independent experiments corresponding to the peptides detected following in in vitro digestion of the GPIPEPRNMDFCQKNPQP peptide in the absence or presence of the human MT2-MMP recombinant catalytic domain (rhMT2). (D) Western blot analysis of lysates recovered from MDCK transfectants cultured with different calcium concentrations. Results are representative of two independent experiments. (E) Representative orthogonal x–z views of confocal images for polarized MDCK transfectants co-immunostained for HA (MT2-MMP, green), E-cadherin (red) and nuclei (Hoechst, blue).

Journal: Journal of Cell Science

Article Title: E-cadherin cleavage by MT2-MMP regulates apical junctional signaling and epithelial homeostasis in the intestine

doi: 10.1242/jcs.203687

Figure Lengend Snippet: E-cadherin is cleaved by MT2-MMP after N445 in the EC5 loop. (A) In silico model of canine E-cadherin (green)/human MT2-MMP (blue) interactions in cis association at the plasma membrane; the catalytic MT2-MMP center and the E-cadherin peptide, GPIPEPRNMDFCQKNPQP, are shown in orange and red, respectively. (B) Scheme of E-cadherin structure with the peptide containing the predicted cleavage sites after N445 and N459 in the EC5 loop. (C) Representative extracted ion chromatograms of 3 independent experiments corresponding to the peptides detected following in in vitro digestion of the GPIPEPRNMDFCQKNPQP peptide in the absence or presence of the human MT2-MMP recombinant catalytic domain (rhMT2). (D) Western blot analysis of lysates recovered from MDCK transfectants cultured with different calcium concentrations. Results are representative of two independent experiments. (E) Representative orthogonal x–z views of confocal images for polarized MDCK transfectants co-immunostained for HA (MT2-MMP, green), E-cadherin (red) and nuclei (Hoechst, blue).

Article Snippet: Antibodies Antibodies used were against β-actin (Sigma-Aldrich, A5441), GST (Thermo Fisher Scientific, A5800), HA (Covance, MMS-101P), MT1-MMP (LEM2/63; Gálvez et al., 2001 ), MT2-MMP (R&D Systems, MAB9161; and a rabbit polyclonal antibody generated by our group at CNB, Madrid, Spain, directed against 16 aa of hMT2 DEPWTFSSTDLHGNNL), Tubulin (Sigma, T6074), pSrc (Cell Signaling, 2101), ZO-1 (Thermo Fisher Scientific, 40-2300), E-cadherin (Cell Signaling, 3195 and BD Biosciences, 610181), Hoechst 3342 (Thermo Fisher Scientific), Ki67 (Abcam, ab16667), Phalloidin 647 (Thermo Fisher Scientific, {"type":"entrez-nucleotide","attrs":{"text":"A22287","term_id":"641467"}} A22287 ), Myosin IIB (Santa Cruz Biotechnology, sc-15370), β-catenin (BD Biosciences, 610153), Rho-GDI (Santa Cruz Biotechnology, sc-360), Ezrin (Upstate, 07-130), EEA1 (Santa Cruz Biotechnology, sc-6415), TfR (Invitrogen, H68.4), HGS (Abcam, ab72053), TSG101 (Abcam, ab30871).

Techniques: In Silico, Clinical Proteomics, Membrane, In Vitro, Recombinant, Western Blot, Cell Culture

MT2-MMP disrupts apical E-cadherin-mediated signals. (A) Orthogonal x–z projections of 3D confocal image stacks are shown of polarized MDCK transfectants stained for F-actin (Phalloidin, green), myosin IIB (red), and nuclei (Hoechst, blue). (B) Orthogonal x–z projections of 3D confocal image stacks are shown of polarized MDCK transfectants (mock and MT2-MMP) in the presence of 4-HAP (500 nM) or vehicle (DMSO) for 72 h and stained for F-actin (phalloidin, green), myosin IIB (red), and nuclei (Hoechst, blue). (C) Quantification of apical/total MFI of myosin IIB in polarized MDCK transfectants treated with 4-HAP (500 nM) or vehicle (DMSO); n=5 independent experiments. (D) Quantification of cell circularity in MDCK cells presented in C. 25 cells per field were counted in 2 images per condition in 6 independent experiments. (E) Quantification of the number of apical events on polarized MDCK cells presented in panel B. 10 fields were counted per condition in n=4 independent experiments. Difference between mock DMSO1 and MT2 FL1, and mock DMSO2 and MT2 FL2 were significant with P<0.0001 and P<0.05, respectively. Data are represented as mean±s.e.m. and were tested by one-way ANOVA followed by Sidak post-test. *P<0.05, **P<0.01, ****P<0.0001; ns, not significant.

Journal: Journal of Cell Science

Article Title: E-cadherin cleavage by MT2-MMP regulates apical junctional signaling and epithelial homeostasis in the intestine

doi: 10.1242/jcs.203687

Figure Lengend Snippet: MT2-MMP disrupts apical E-cadherin-mediated signals. (A) Orthogonal x–z projections of 3D confocal image stacks are shown of polarized MDCK transfectants stained for F-actin (Phalloidin, green), myosin IIB (red), and nuclei (Hoechst, blue). (B) Orthogonal x–z projections of 3D confocal image stacks are shown of polarized MDCK transfectants (mock and MT2-MMP) in the presence of 4-HAP (500 nM) or vehicle (DMSO) for 72 h and stained for F-actin (phalloidin, green), myosin IIB (red), and nuclei (Hoechst, blue). (C) Quantification of apical/total MFI of myosin IIB in polarized MDCK transfectants treated with 4-HAP (500 nM) or vehicle (DMSO); n=5 independent experiments. (D) Quantification of cell circularity in MDCK cells presented in C. 25 cells per field were counted in 2 images per condition in 6 independent experiments. (E) Quantification of the number of apical events on polarized MDCK cells presented in panel B. 10 fields were counted per condition in n=4 independent experiments. Difference between mock DMSO1 and MT2 FL1, and mock DMSO2 and MT2 FL2 were significant with P<0.0001 and P<0.05, respectively. Data are represented as mean±s.e.m. and were tested by one-way ANOVA followed by Sidak post-test. *P<0.05, **P<0.01, ****P<0.0001; ns, not significant.

Article Snippet: Antibodies Antibodies used were against β-actin (Sigma-Aldrich, A5441), GST (Thermo Fisher Scientific, A5800), HA (Covance, MMS-101P), MT1-MMP (LEM2/63; Gálvez et al., 2001 ), MT2-MMP (R&D Systems, MAB9161; and a rabbit polyclonal antibody generated by our group at CNB, Madrid, Spain, directed against 16 aa of hMT2 DEPWTFSSTDLHGNNL), Tubulin (Sigma, T6074), pSrc (Cell Signaling, 2101), ZO-1 (Thermo Fisher Scientific, 40-2300), E-cadherin (Cell Signaling, 3195 and BD Biosciences, 610181), Hoechst 3342 (Thermo Fisher Scientific), Ki67 (Abcam, ab16667), Phalloidin 647 (Thermo Fisher Scientific, {"type":"entrez-nucleotide","attrs":{"text":"A22287","term_id":"641467"}} A22287 ), Myosin IIB (Santa Cruz Biotechnology, sc-15370), β-catenin (BD Biosciences, 610153), Rho-GDI (Santa Cruz Biotechnology, sc-360), Ezrin (Upstate, 07-130), EEA1 (Santa Cruz Biotechnology, sc-6415), TfR (Invitrogen, H68.4), HGS (Abcam, ab72053), TSG101 (Abcam, ab30871).

Techniques: Staining

Mislocalization of pSrc in polarized MT2-MMP-MDCK cells contributes to apical cell accumulation. (A) Percentage of cells in G0/G1, S, and G2/M phases of the cell cycle analyzed by flow cytometry in propidium-iodide-stained MDCK transfectants after 72 h of serum deprivation. Means±s.e.m. are shown for 3 independent experiments. (B) Orthogonal x–z projections of 3D confocal image stacks of polarized MDCK transfectants (mock and MT2-MMP) stained for F-actin (Phalloidin, green), pSrc (red), and nuclei (Hoechst, blue). Representative peak intensity profiles are shown on the right for pSrc (red) and F-actin (green). (C) Bar graphs show the apical (left) and junctional (right) pSrc intensity, relative to total mean fluorescence intensity (MFI) in 6 independent experiments. (D) Number of apical events occurring in polarized MDCK cells treated with PP2 or vehicle (DMSO). 10 fields were counted per condition in 3 independent experiments. Differences between mock DMSO1 and MT2 FL1, and mock DMSO2 and MT2 FL2 were significant with P<0.001 and P<0.01, respectively. (E) Representative confocal images of 3D cysts formed by MDCK transfectants in Matrigel and stained for pSrc (green), F-actin (white), E-cadherin (red), and nuclei (Hoechst, blue). (F) Quantification of the percentage of lumenized cysts. Data are represented as the mean±s.e.m. and were tested by two-way ANOVA followed by Dunnett's post-test in A, two-tailed Welch-test comparison was used in B, and one-way ANOVA followed by Sidak post-test was used in C. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; ns, not significant.

Journal: Journal of Cell Science

Article Title: E-cadherin cleavage by MT2-MMP regulates apical junctional signaling and epithelial homeostasis in the intestine

doi: 10.1242/jcs.203687

Figure Lengend Snippet: Mislocalization of pSrc in polarized MT2-MMP-MDCK cells contributes to apical cell accumulation. (A) Percentage of cells in G0/G1, S, and G2/M phases of the cell cycle analyzed by flow cytometry in propidium-iodide-stained MDCK transfectants after 72 h of serum deprivation. Means±s.e.m. are shown for 3 independent experiments. (B) Orthogonal x–z projections of 3D confocal image stacks of polarized MDCK transfectants (mock and MT2-MMP) stained for F-actin (Phalloidin, green), pSrc (red), and nuclei (Hoechst, blue). Representative peak intensity profiles are shown on the right for pSrc (red) and F-actin (green). (C) Bar graphs show the apical (left) and junctional (right) pSrc intensity, relative to total mean fluorescence intensity (MFI) in 6 independent experiments. (D) Number of apical events occurring in polarized MDCK cells treated with PP2 or vehicle (DMSO). 10 fields were counted per condition in 3 independent experiments. Differences between mock DMSO1 and MT2 FL1, and mock DMSO2 and MT2 FL2 were significant with P<0.001 and P<0.01, respectively. (E) Representative confocal images of 3D cysts formed by MDCK transfectants in Matrigel and stained for pSrc (green), F-actin (white), E-cadherin (red), and nuclei (Hoechst, blue). (F) Quantification of the percentage of lumenized cysts. Data are represented as the mean±s.e.m. and were tested by two-way ANOVA followed by Dunnett's post-test in A, two-tailed Welch-test comparison was used in B, and one-way ANOVA followed by Sidak post-test was used in C. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; ns, not significant.

Article Snippet: Antibodies Antibodies used were against β-actin (Sigma-Aldrich, A5441), GST (Thermo Fisher Scientific, A5800), HA (Covance, MMS-101P), MT1-MMP (LEM2/63; Gálvez et al., 2001 ), MT2-MMP (R&D Systems, MAB9161; and a rabbit polyclonal antibody generated by our group at CNB, Madrid, Spain, directed against 16 aa of hMT2 DEPWTFSSTDLHGNNL), Tubulin (Sigma, T6074), pSrc (Cell Signaling, 2101), ZO-1 (Thermo Fisher Scientific, 40-2300), E-cadherin (Cell Signaling, 3195 and BD Biosciences, 610181), Hoechst 3342 (Thermo Fisher Scientific), Ki67 (Abcam, ab16667), Phalloidin 647 (Thermo Fisher Scientific, {"type":"entrez-nucleotide","attrs":{"text":"A22287","term_id":"641467"}} A22287 ), Myosin IIB (Santa Cruz Biotechnology, sc-15370), β-catenin (BD Biosciences, 610153), Rho-GDI (Santa Cruz Biotechnology, sc-360), Ezrin (Upstate, 07-130), EEA1 (Santa Cruz Biotechnology, sc-6415), TfR (Invitrogen, H68.4), HGS (Abcam, ab72053), TSG101 (Abcam, ab30871).

Techniques: Flow Cytometry, Staining, Fluorescence, Two Tailed Test, Comparison

MT2-MMP deficiency alters junctional E-cadherin and leads to decreased 3D colon organoid formation ex vivo and smaller crypts in vivo. (A) Representative confocal images of MT2-MMP-silenced colon organoids stained for MT2-MMP (green; top). Graph shows the normalized MT2-MMP mean fluorescence intensity (MFI) in stained organoids 72 h after siRNA transfection (bottom), n=6 images per condition from 3 independent experiments; Mmp15 mRNA levels decreased ∼20% in silenced organoids. (B) Bright-field microscopy images of MT2-MMP-silenced colon organoids. Bar graph shows the percentage of organoid generation efficiency 48 h after siRNA transfection (right) in 3 independent experiments. (C) Representative confocal images of MT2-MMP-silenced colon organoids stained for nuclei (Hoechst/Ho, blue), F-actin (red), E-cadherin (green) and β-catenin (white); magnified views of E-cadherin and β-catenin staining are shown in insets. (D) Representative confocal images of colonic tissues recovered from wild-type or MT2-MMP-null mice, and stained for Ki67 (red) and nuclei (Hoechst, blue). On the right, graph shows the percentage of Ki67-positive cells per crypt. 9–15 crypts were quantified per condition in 3 images taken from 2 mice per genotype (top). (E) Quantification of the cumulative frequency of crypt length (left) and width (right). Data are represented as mean±s.e.m. and were tested by unpaired Student's t-test in A and B and by two-tailed Welch-test comparison in D and E. *P<0.05, **P<0.01, ***P<0.001; ns, not significant.

Journal: Journal of Cell Science

Article Title: E-cadherin cleavage by MT2-MMP regulates apical junctional signaling and epithelial homeostasis in the intestine

doi: 10.1242/jcs.203687

Figure Lengend Snippet: MT2-MMP deficiency alters junctional E-cadherin and leads to decreased 3D colon organoid formation ex vivo and smaller crypts in vivo. (A) Representative confocal images of MT2-MMP-silenced colon organoids stained for MT2-MMP (green; top). Graph shows the normalized MT2-MMP mean fluorescence intensity (MFI) in stained organoids 72 h after siRNA transfection (bottom), n=6 images per condition from 3 independent experiments; Mmp15 mRNA levels decreased ∼20% in silenced organoids. (B) Bright-field microscopy images of MT2-MMP-silenced colon organoids. Bar graph shows the percentage of organoid generation efficiency 48 h after siRNA transfection (right) in 3 independent experiments. (C) Representative confocal images of MT2-MMP-silenced colon organoids stained for nuclei (Hoechst/Ho, blue), F-actin (red), E-cadherin (green) and β-catenin (white); magnified views of E-cadherin and β-catenin staining are shown in insets. (D) Representative confocal images of colonic tissues recovered from wild-type or MT2-MMP-null mice, and stained for Ki67 (red) and nuclei (Hoechst, blue). On the right, graph shows the percentage of Ki67-positive cells per crypt. 9–15 crypts were quantified per condition in 3 images taken from 2 mice per genotype (top). (E) Quantification of the cumulative frequency of crypt length (left) and width (right). Data are represented as mean±s.e.m. and were tested by unpaired Student's t-test in A and B and by two-tailed Welch-test comparison in D and E. *P<0.05, **P<0.01, ***P<0.001; ns, not significant.

Article Snippet: Antibodies Antibodies used were against β-actin (Sigma-Aldrich, A5441), GST (Thermo Fisher Scientific, A5800), HA (Covance, MMS-101P), MT1-MMP (LEM2/63; Gálvez et al., 2001 ), MT2-MMP (R&D Systems, MAB9161; and a rabbit polyclonal antibody generated by our group at CNB, Madrid, Spain, directed against 16 aa of hMT2 DEPWTFSSTDLHGNNL), Tubulin (Sigma, T6074), pSrc (Cell Signaling, 2101), ZO-1 (Thermo Fisher Scientific, 40-2300), E-cadherin (Cell Signaling, 3195 and BD Biosciences, 610181), Hoechst 3342 (Thermo Fisher Scientific), Ki67 (Abcam, ab16667), Phalloidin 647 (Thermo Fisher Scientific, {"type":"entrez-nucleotide","attrs":{"text":"A22287","term_id":"641467"}} A22287 ), Myosin IIB (Santa Cruz Biotechnology, sc-15370), β-catenin (BD Biosciences, 610153), Rho-GDI (Santa Cruz Biotechnology, sc-360), Ezrin (Upstate, 07-130), EEA1 (Santa Cruz Biotechnology, sc-6415), TfR (Invitrogen, H68.4), HGS (Abcam, ab72053), TSG101 (Abcam, ab30871).

Techniques: Ex Vivo, In Vivo, Staining, Fluorescence, Transfection, Microscopy, Two Tailed Test, Comparison

Fig. 3. The cytotoxicity of Cd and intracellular Cd accumulation with or without GOs pre-treatment. (a) The cellular viability of BEAS-2B cells detected by CCK8 assay after being treated to Cd (1, 5, 10, 20, 25, 30 and 50 μM) for 24 h (n = 5, * indicate P < 0.05, compared to the untreated group). (b) The heat map of the cell viability inspected by CCK8 assay for BEAS- 2B cells after being treated to Cd (1, 5, 10, 20, 25, 30 and 50 μM) for 24 h with or without pre-treatment of P-GO, A-GO, or G-GO at the dose of 10 μg/mL, respectively (n = 5). (c) Intracellular Cd mass quan tification by ICP-MS. BEAS-2B cells were pre-treated with P-GO, A-GO, G-GO at 10 μg/mL for 24 h, and then exposed to CdCl2 at 10 μM for 24 h (n = 5), *P < 0.05. (d) Western blot analysis of MT1M and MT2A protein expression levels in BEAS-2B cells exposed to either GOs (10 μg/mL) or Cd (10 μM) or a combina tion of both (GOs + Cd) for 24 h.

Journal: Environmental pollution (Barking, Essex : 1987)

Article Title: Biotransformation of graphene oxide within lung fluids could intensify its synergistic biotoxicity effect with cadmium by inhibiting cellular efflux of cadmium.

doi: 10.1016/j.envpol.2022.119421

Figure Lengend Snippet: Fig. 3. The cytotoxicity of Cd and intracellular Cd accumulation with or without GOs pre-treatment. (a) The cellular viability of BEAS-2B cells detected by CCK8 assay after being treated to Cd (1, 5, 10, 20, 25, 30 and 50 μM) for 24 h (n = 5, * indicate P < 0.05, compared to the untreated group). (b) The heat map of the cell viability inspected by CCK8 assay for BEAS- 2B cells after being treated to Cd (1, 5, 10, 20, 25, 30 and 50 μM) for 24 h with or without pre-treatment of P-GO, A-GO, or G-GO at the dose of 10 μg/mL, respectively (n = 5). (c) Intracellular Cd mass quan tification by ICP-MS. BEAS-2B cells were pre-treated with P-GO, A-GO, G-GO at 10 μg/mL for 24 h, and then exposed to CdCl2 at 10 μM for 24 h (n = 5), *P < 0.05. (d) Western blot analysis of MT1M and MT2A protein expression levels in BEAS-2B cells exposed to either GOs (10 μg/mL) or Cd (10 μM) or a combina tion of both (GOs + Cd) for 24 h.

Article Snippet: Antibodies (Abs) used were as follows: anti-NRF2 Ab (1:2000 dilution, Proteintech, USA), anti-SOD1 Ab (1:5000 dilution, Proteintech, USA), anti-SOD2 Ab (1:3000 dilution, Proteintech, USA), anti-MT1M (1:500 dilution, Proteintech, USA), anti-MT2A Ab (1:2000 dilution, Affinity, USA), anti-Caspase-8 Ab (1:5000 dilution, Proteintech, USA), anti-Bax Ab (1:5000 dilution, Proteintech, USA), anti-Bcl-2 Ab (1:5000 dilution, Proteintech, USA), anti-RIP1 Ab (1:2500 dilution, Proteintech, USA), anti-RIP3 Ab (1:2500 dilution, Proteintech, USA), anti-ABCB1 Ab (1:5000 dilution, Proteintech, USA), anti-ABCC1 Ab (1:2500 dilution, Proteintech, USA), anti-ABCG2 Ab (1:1000 dilution, Proteintech, USA).

Techniques: CCK-8 Assay, Western Blot, Expressing

HEK293 cells transfected with MT1-MMP cDNA invade 3D collagen gels in response to LPA . (A) Tumor cell lysates were prepared for Western blot analysis. Lysates were probed for MT1-MMP to assess protein expression in the four tumor cell lines. Lysates were probed for Actin as a loading control. (B) HEK293 cells were transfected with the pAdTrack-CMV plasmid as a control, or plasmids encoding MT1-MMP, MT2-MMP, or MT3-MMP cDNA 24 hours prior to placement in invasion assays. Cells were allowed to invade 2.0 mg/ml collagen gels in the presence or absence of 1 μM LPA. Data are expressed as mean numbers of invading cells per HPF (20×) (± S.D.) from a minimum of 20 fields. (C) Lysates from HEK293 cells transfected with cDNAs encoding the designated genes were prepared for Western blot analysis and probed for GFP, MT1-MMP, MT2-MMP, MT3-MMP, or Actin as a loading control. TRK = pAdTrack-CMV, MT1 = MT1-MMP, MT2 = MT2-MMP, MT3 = MT3-MMP.

Journal: Molecular Cancer

Article Title: Tumor cell invasion of collagen matrices requires coordinate lipid agonist-induced G-protein and membrane-type matrix metalloproteinase-1-dependent signaling

doi: 10.1186/1476-4598-5-69

Figure Lengend Snippet: HEK293 cells transfected with MT1-MMP cDNA invade 3D collagen gels in response to LPA . (A) Tumor cell lysates were prepared for Western blot analysis. Lysates were probed for MT1-MMP to assess protein expression in the four tumor cell lines. Lysates were probed for Actin as a loading control. (B) HEK293 cells were transfected with the pAdTrack-CMV plasmid as a control, or plasmids encoding MT1-MMP, MT2-MMP, or MT3-MMP cDNA 24 hours prior to placement in invasion assays. Cells were allowed to invade 2.0 mg/ml collagen gels in the presence or absence of 1 μM LPA. Data are expressed as mean numbers of invading cells per HPF (20×) (± S.D.) from a minimum of 20 fields. (C) Lysates from HEK293 cells transfected with cDNAs encoding the designated genes were prepared for Western blot analysis and probed for GFP, MT1-MMP, MT2-MMP, MT3-MMP, or Actin as a loading control. TRK = pAdTrack-CMV, MT1 = MT1-MMP, MT2 = MT2-MMP, MT3 = MT3-MMP.

Article Snippet: Human MT1-MMP (AF918, R&D Systems, Minneapolis, MN), MT3-MMP (RP1-MMP-16, Triple Point Biologics, Forest Grove, OR), and Rac1 (ARC01, Cytoskeleton, Denver, CO), and monoclonal antibodies directed against human MT2-MMP (MAB916, R&D Systems), Lamin A/C (MAB3211, Chemicon Corp), Actin (JLA-20, Calbiochem, San Diego, CA), RhoA (ARH01, Cytoskeleton), and Cdc42 (610929, BD Tansduction, San Jose, CA) were used for Western blot analysis as described previously [ ]. si GENOME SMART pool ® reagents were products of Dharmacon (Lafayette, CO).

Techniques: Transfection, Western Blot, Expressing, Control, Plasmid Preparation

FIGURE 5 | Representative western blots showing (A) melatonin receptor-1 (MT1) (37 kDa), and −2 (MT2) (40 kDa), and RORα (59 kDa) bands in control, prenatal dexamethasone exposure (DEX), postnatal high-fat diet (HF), prenatal DEX plus postnatal (DEX+HF), and melatonin treatment (DEX+HF+M) offspring rats at 16 weeks of age. Relative abundance of renal cortical (B) MT1, (C) MT2, and (D) RORα as quantified. (E) Effect of DEX, HF, and melatonin on renal melatonin level. N = 6/group. *P < 0.05 vs. control; #P < 0.05 vs. DEX; ‡P < 0.05 vs. DEX+HF.

Journal: Frontiers in physiology

Article Title: Maternal Melatonin Therapy Rescues Prenatal Dexamethasone and Postnatal High-Fat Diet Induced Programmed Hypertension in Male Rat Offspring.

doi: 10.3389/fphys.2015.00377

Figure Lengend Snippet: FIGURE 5 | Representative western blots showing (A) melatonin receptor-1 (MT1) (37 kDa), and −2 (MT2) (40 kDa), and RORα (59 kDa) bands in control, prenatal dexamethasone exposure (DEX), postnatal high-fat diet (HF), prenatal DEX plus postnatal (DEX+HF), and melatonin treatment (DEX+HF+M) offspring rats at 16 weeks of age. Relative abundance of renal cortical (B) MT1, (C) MT2, and (D) RORα as quantified. (E) Effect of DEX, HF, and melatonin on renal melatonin level. N = 6/group. *P < 0.05 vs. control; #P < 0.05 vs. DEX; ‡P < 0.05 vs. DEX+HF.

Article Snippet: We used the following antibodies: a goat anti-rat MT1 antibody (1:1000, overnight incubation; Santa Cruz Biotechnology, Santa Cruz, CA, USA); a rabbit anti-rat MT2 antibody (1:1000, overnight incubation; Biorbyt, AllBio Science Inc., Taichung, Taiwan); and a rabbit anti-rat RORα antibody (1:2000, overnight incubation; Proteintech Group, Inc., Chicago, IL, USA).

Techniques: Western Blot, Control

XBP1 expression in macrophages is increased and positively correlates with IR-induced liver injury in patients. Liver tissues and macrophages were harvested from 25 patients undergoing PH. ( A-D ) Prehepatectomy/posthepatectomy XBP1s expression profiles in liver tissues and macrophages (representative 6 cases) through Western blotting assay. ( B, C ) Expressions of XBP1 mRNA in liver tissues and in liver macrophages. ( E ) Dual-immunofluorescence staining for XBP1 ( green ) and CD68 ( red ) colocalization in human liver tissues, which were also subjected to HE and TUNEL analysis. ( F ) Twenty-five patients were divided into the low (n = 12) and high (n = 13) groups using the postoperative median XBP1/GAPDH ratio as the threshold. ( G ) The ratio of posthepatectomy XBP1/GAPDH positively correlated with serum ALT and AST levels at POD1. ( H ) Serum ALT and AST levels were measured at POD1 in both low and high groups. ∗ P < .05 by Student t test.

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: XBP1 Facilitating NF-κB-p65 Nuclear Translocation Promotes Macrophage-Originated Sterile Inflammation Via Regulating MT2 Transcription in the Ischemia/Reperfusion Liver

doi: 10.1016/j.jcmgh.2024.101402

Figure Lengend Snippet: XBP1 expression in macrophages is increased and positively correlates with IR-induced liver injury in patients. Liver tissues and macrophages were harvested from 25 patients undergoing PH. ( A-D ) Prehepatectomy/posthepatectomy XBP1s expression profiles in liver tissues and macrophages (representative 6 cases) through Western blotting assay. ( B, C ) Expressions of XBP1 mRNA in liver tissues and in liver macrophages. ( E ) Dual-immunofluorescence staining for XBP1 ( green ) and CD68 ( red ) colocalization in human liver tissues, which were also subjected to HE and TUNEL analysis. ( F ) Twenty-five patients were divided into the low (n = 12) and high (n = 13) groups using the postoperative median XBP1/GAPDH ratio as the threshold. ( G ) The ratio of posthepatectomy XBP1/GAPDH positively correlated with serum ALT and AST levels at POD1. ( H ) Serum ALT and AST levels were measured at POD1 in both low and high groups. ∗ P < .05 by Student t test.

Article Snippet: The antibodies used were as follows: XBP1s, Bcl2, Bcl-XL, C-Caspase3, NF-κB p65, P-NF-κB p65, GAPDH (Cell Signaling Technology), and MT2 (Abclonal, WuHan, China).

Techniques: Expressing, Western Blot, Immunofluorescence, Staining, TUNEL Assay

Myeloid-specific XBP1 deficiency attenuates IR-induced hepatocellular injury. The IRI model mice were subjected to 90-minute ischemia, followed by 6-hour reperfusion. ( A, B ) XBP1s expression profiles in IR liver tissues and nonparenchymal cells, and their relative quantitation (fold change) via Western blotting assay. ( C ) Serum ALT and AST levels in the sham and IR groups were measured. ( D ) Generation flow chart of XBP1 M-KO mice. ( E ) Comparison of XBP1s expression in XBP1 FL/FL and XBP M-KO mice under normal conditions (Sham) and after I/R injury. ( F ) Representative HE staining of ischemic liver tissues, with the scale of 200 μm. ( G ) Liver damage was evaluated by Suzuki’s histologic scores. ( H, I ) ROS-sensing dye DCFH-DA staining and quantitation. ( J, K ) TUNEL staining of liver tissues and positive cell percentage. ( L ) Bcl-xl, Bcl-2, C-caspase3, P-NF-kB-p65, and NF-kB-p65 profiles in liver tissues based on Western blotting assay. n = 6, ∗ P < .05 by Student t test.

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: XBP1 Facilitating NF-κB-p65 Nuclear Translocation Promotes Macrophage-Originated Sterile Inflammation Via Regulating MT2 Transcription in the Ischemia/Reperfusion Liver

doi: 10.1016/j.jcmgh.2024.101402

Figure Lengend Snippet: Myeloid-specific XBP1 deficiency attenuates IR-induced hepatocellular injury. The IRI model mice were subjected to 90-minute ischemia, followed by 6-hour reperfusion. ( A, B ) XBP1s expression profiles in IR liver tissues and nonparenchymal cells, and their relative quantitation (fold change) via Western blotting assay. ( C ) Serum ALT and AST levels in the sham and IR groups were measured. ( D ) Generation flow chart of XBP1 M-KO mice. ( E ) Comparison of XBP1s expression in XBP1 FL/FL and XBP M-KO mice under normal conditions (Sham) and after I/R injury. ( F ) Representative HE staining of ischemic liver tissues, with the scale of 200 μm. ( G ) Liver damage was evaluated by Suzuki’s histologic scores. ( H, I ) ROS-sensing dye DCFH-DA staining and quantitation. ( J, K ) TUNEL staining of liver tissues and positive cell percentage. ( L ) Bcl-xl, Bcl-2, C-caspase3, P-NF-kB-p65, and NF-kB-p65 profiles in liver tissues based on Western blotting assay. n = 6, ∗ P < .05 by Student t test.

Article Snippet: The antibodies used were as follows: XBP1s, Bcl2, Bcl-XL, C-Caspase3, NF-κB p65, P-NF-κB p65, GAPDH (Cell Signaling Technology), and MT2 (Abclonal, WuHan, China).

Techniques: Expressing, Quantitation Assay, Western Blot, Comparison, Staining, TUNEL Assay

Myeloid-specific XBP1 deficiency alleviates sterile inflammation. ( A ) BMMs were cocultured with primary hepatocytes rendered anoxic using an anaerobic cell incubator. ( B ) ALT and AST levels were measured in the supernatant from hepatocytes in H/R system. ( C ) The ALT and AST levels detected in the cocultured H/R model from XBP1 FL/FL and XBP1 M-KO mice groups. ( D, E ) XBP1s expression profiles in XBP1 FL/FL BMMs in the control and H/R cocultured system, and their quantitation (fold change) based on Western blotting assay. ( F, G ) XBP1s expression profiles in XBP1 FL/FL and XBP1 M-KO BMMs in H/R cocultured system using Western blotting assay. ( H ) Immunofluorescence staining for XBP1s ( green ) in cocultured XBP1 FL/FL BMMs. ( I ) XBP1s expression in cytoplasm and nucleus of cocultured XBP1 FL/FL BMMs using Western blotting assay. ( J ) Detection of TNF-α, IL1β, IL6, iNOS, and IL10 in H/R cocultured BMMs by qRT-PCR. ( K, L ) NLRP3, P-NF-κBp65, and NF-κBp65 protein expression levels in H/R cocultured BMMs and their quantitation (fold change) using Western blotting assay. ( M, N ) Migratory abilities of BMMs cocultured for 24 hours based on the Transwell assay. n = 6, ∗ P < .05 by Student t test.

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: XBP1 Facilitating NF-κB-p65 Nuclear Translocation Promotes Macrophage-Originated Sterile Inflammation Via Regulating MT2 Transcription in the Ischemia/Reperfusion Liver

doi: 10.1016/j.jcmgh.2024.101402

Figure Lengend Snippet: Myeloid-specific XBP1 deficiency alleviates sterile inflammation. ( A ) BMMs were cocultured with primary hepatocytes rendered anoxic using an anaerobic cell incubator. ( B ) ALT and AST levels were measured in the supernatant from hepatocytes in H/R system. ( C ) The ALT and AST levels detected in the cocultured H/R model from XBP1 FL/FL and XBP1 M-KO mice groups. ( D, E ) XBP1s expression profiles in XBP1 FL/FL BMMs in the control and H/R cocultured system, and their quantitation (fold change) based on Western blotting assay. ( F, G ) XBP1s expression profiles in XBP1 FL/FL and XBP1 M-KO BMMs in H/R cocultured system using Western blotting assay. ( H ) Immunofluorescence staining for XBP1s ( green ) in cocultured XBP1 FL/FL BMMs. ( I ) XBP1s expression in cytoplasm and nucleus of cocultured XBP1 FL/FL BMMs using Western blotting assay. ( J ) Detection of TNF-α, IL1β, IL6, iNOS, and IL10 in H/R cocultured BMMs by qRT-PCR. ( K, L ) NLRP3, P-NF-κBp65, and NF-κBp65 protein expression levels in H/R cocultured BMMs and their quantitation (fold change) using Western blotting assay. ( M, N ) Migratory abilities of BMMs cocultured for 24 hours based on the Transwell assay. n = 6, ∗ P < .05 by Student t test.

Article Snippet: The antibodies used were as follows: XBP1s, Bcl2, Bcl-XL, C-Caspase3, NF-κB p65, P-NF-κB p65, GAPDH (Cell Signaling Technology), and MT2 (Abclonal, WuHan, China).

Techniques: Sterility, Expressing, Control, Quantitation Assay, Western Blot, Immunofluorescence, Staining, Quantitative RT-PCR, Transwell Assay

STAT phosphorylation and IFN‐I dependent NK cell activation following reovirus treatment. (a) STAT phosphorylation in CD56 bright and CD56 dim NK cells (detected by intracellular staining and flow cytometry) in PBMC cultured without virus (untreated; black line) or with 1 MOI reovirus (purple line), for 8, 24 and 48 h. Graphs show mean MFI and standard deviation from three donors. Data were analysed by two‐way repeated‐measures ANOVA, followed by Sidak multiple comparisons test. * p < 0·05 ** p < 0·01. (b) NK cell activation by reovirus is IFN‐I dependent. The flow chart shows the approach taken; PBMC (from one donor) were left untreated or treated with reovirus for 24 h and the conditioned media (CM) filtered to remove viruses. CM was added to purified NK cells in the presence of an IFN‐I blocking antibody cocktail (IFN block), a control blocking cocktail (control block) or no added antibody (no block). CM from untreated PBMC was used a control. After 48 h, the NK cell surface expression of CD69 and tetherin was measured by flow cytometry. Data is from control CM or CM from reovirus‐treated PBMC from a single donor, applied to three NK cell donors. The y ‐axes show the percentage of CD69 expressing cells (top panel) or the fold change in MFI of tetherin relative to control CM and no added antibody treatment (bottom panel), due to constitutive low‐level expression of this molecule on unstimulated NK cells . Differences between mean percentage positive values for CD69, or mean fold change MFI for tetherin, were analysed by two‐way repeated‐measures ANOVA, followed by Sidak multiple comparisons test. * p < 0·05 ** p < 0·01

Journal: Immunology

Article Title: Oncolytic virus treatment differentially affects the CD56 dim and CD56 bright NK cell subsets in vivo and regulates a spectrum of human NK cell activity

doi: 10.1111/imm.13453

Figure Lengend Snippet: STAT phosphorylation and IFN‐I dependent NK cell activation following reovirus treatment. (a) STAT phosphorylation in CD56 bright and CD56 dim NK cells (detected by intracellular staining and flow cytometry) in PBMC cultured without virus (untreated; black line) or with 1 MOI reovirus (purple line), for 8, 24 and 48 h. Graphs show mean MFI and standard deviation from three donors. Data were analysed by two‐way repeated‐measures ANOVA, followed by Sidak multiple comparisons test. * p < 0·05 ** p < 0·01. (b) NK cell activation by reovirus is IFN‐I dependent. The flow chart shows the approach taken; PBMC (from one donor) were left untreated or treated with reovirus for 24 h and the conditioned media (CM) filtered to remove viruses. CM was added to purified NK cells in the presence of an IFN‐I blocking antibody cocktail (IFN block), a control blocking cocktail (control block) or no added antibody (no block). CM from untreated PBMC was used a control. After 48 h, the NK cell surface expression of CD69 and tetherin was measured by flow cytometry. Data is from control CM or CM from reovirus‐treated PBMC from a single donor, applied to three NK cell donors. The y ‐axes show the percentage of CD69 expressing cells (top panel) or the fold change in MFI of tetherin relative to control CM and no added antibody treatment (bottom panel), due to constitutive low‐level expression of this molecule on unstimulated NK cells . Differences between mean percentage positive values for CD69, or mean fold change MFI for tetherin, were analysed by two‐way repeated‐measures ANOVA, followed by Sidak multiple comparisons test. * p < 0·05 ** p < 0·01

Article Snippet: For IFN‐I ELISA, 96 well plates were coated with a mixture of antibodies against IFN‐α (Mabtech, MT1/3/5) overnight at 4°C, and blocked with PBS supplemented with 10% FCS.

Techniques: Phospho-proteomics, Activation Assay, Staining, Flow Cytometry, Cell Culture, Virus, Standard Deviation, Purification, Blocking Assay, Control, Expressing

Gene expression profiling of NK cells following reovirus treatment. (a) Summary of the approach. PBMC from five healthy donors were treated at an MOI of 1 with reovirus (or left untreated). After 48 h, NK cells were purified using immunomagnetic selection and a small aliquot of cells was removed for flow cytometry to assess CD69 induction (Figure ). The remainder of the NK cells were used in the gene expression profiling. (b) Differentially expressed genes in total human NK cells following reovirus treatment. Genes that were up (green columns) or downregulated (red columns) together with examples and fold change are indicated. Data underlying this graph is shown in Table . (c) Gene set enrichment analysis (GSEA). The left panel shows the top 10 pathways identified by GSEA of differentially expressed genes. Data were generated using Enrichr and the output from Reactome 2016; the adjusted p ‐value of enrichment is shown. Full data is provided in Table . The right panel shows the top 10 transcription factors associated with differentially expressed genes. Data were generated using Enrichr and the output from ENCODE and ChEA Consensus Transcription factors from ChIP‐X; the adjusted p ‐value of enrichment is shown. Full data are provided in Table . (d) Venn diagrams showing the overlap of the differentially expressed genes (DEGs) from NK cells following reovirus treatment (Reo) with the interferon‐stimulated genes listed in Schoggins et al (; S‐Set) or the core mammalian ISGs identified by Shaw et al (; Core m‐ISGs). Overlaps were determined for all reovirus DEGs (left and right panels) or the upregulated genes only (centre panel). (e) Expression of genes from selected pathways. The graph shows the fold change in gene expression in NK cells following reovirus treatment, with genes and pathways indicated. A green circle below the x ‐axis indicates that the gene is induced by IFN‐I in haematopoietic cells, as determined using data from the Interferome database . (f) TRAIL expression by CD56 dim and CD56 bright NK cells following reovirus treatment. The histograms show flow cytometry from a single representative donor, with the different treatments (and isotype control antibody) indicated, along with the NK cell subset analysed via gating. The graphs on the right show the mean and standard deviation of the median fluorescence intensity (MFI), from three separate donors; data were analysed by a repeated‐measures one‐way ANOVA, with Tukey's multiple comparison test; ** p < 0·01

Journal: Immunology

Article Title: Oncolytic virus treatment differentially affects the CD56 dim and CD56 bright NK cell subsets in vivo and regulates a spectrum of human NK cell activity

doi: 10.1111/imm.13453

Figure Lengend Snippet: Gene expression profiling of NK cells following reovirus treatment. (a) Summary of the approach. PBMC from five healthy donors were treated at an MOI of 1 with reovirus (or left untreated). After 48 h, NK cells were purified using immunomagnetic selection and a small aliquot of cells was removed for flow cytometry to assess CD69 induction (Figure ). The remainder of the NK cells were used in the gene expression profiling. (b) Differentially expressed genes in total human NK cells following reovirus treatment. Genes that were up (green columns) or downregulated (red columns) together with examples and fold change are indicated. Data underlying this graph is shown in Table . (c) Gene set enrichment analysis (GSEA). The left panel shows the top 10 pathways identified by GSEA of differentially expressed genes. Data were generated using Enrichr and the output from Reactome 2016; the adjusted p ‐value of enrichment is shown. Full data is provided in Table . The right panel shows the top 10 transcription factors associated with differentially expressed genes. Data were generated using Enrichr and the output from ENCODE and ChEA Consensus Transcription factors from ChIP‐X; the adjusted p ‐value of enrichment is shown. Full data are provided in Table . (d) Venn diagrams showing the overlap of the differentially expressed genes (DEGs) from NK cells following reovirus treatment (Reo) with the interferon‐stimulated genes listed in Schoggins et al (; S‐Set) or the core mammalian ISGs identified by Shaw et al (; Core m‐ISGs). Overlaps were determined for all reovirus DEGs (left and right panels) or the upregulated genes only (centre panel). (e) Expression of genes from selected pathways. The graph shows the fold change in gene expression in NK cells following reovirus treatment, with genes and pathways indicated. A green circle below the x ‐axis indicates that the gene is induced by IFN‐I in haematopoietic cells, as determined using data from the Interferome database . (f) TRAIL expression by CD56 dim and CD56 bright NK cells following reovirus treatment. The histograms show flow cytometry from a single representative donor, with the different treatments (and isotype control antibody) indicated, along with the NK cell subset analysed via gating. The graphs on the right show the mean and standard deviation of the median fluorescence intensity (MFI), from three separate donors; data were analysed by a repeated‐measures one‐way ANOVA, with Tukey's multiple comparison test; ** p < 0·01

Article Snippet: For IFN‐I ELISA, 96 well plates were coated with a mixture of antibodies against IFN‐α (Mabtech, MT1/3/5) overnight at 4°C, and blocked with PBS supplemented with 10% FCS.

Techniques: Gene Expression, Purification, Selection, Flow Cytometry, Generated, Expressing, Control, Standard Deviation, Fluorescence, Comparison

Reovirus‐induced IFN‐I production inhibits IL‐15‐mediated proliferation of NK cells. (a) Summary of the approach; Conditioned media (CM) was collected from reovirus treated (or untreated) PBMC and filtered to remove virions. This CM was applied to fresh PBMC (labelled with CFSE) together with IL‐15, in the presence or absence of IFN‐I blocking antibodies. PBMC were cultured for 5 days and proliferation of CD56 bright NK cells analysed by CFSE content. (b) Inhibition of CD56 bright NK cell proliferation by reovirus‐induced IFN‐I. CFSE analysis of CD56 brigh t NK cells following treatment described in (a). The percentage of cells that have proliferated after 5 days are indicated. These data are from a single donor, representative of three donors. (c) IFN‐I blocks IL‐15 induced S phase. Purified NK cells were cultured with IL‐15 or IL‐15+IFN‐I (all at 100 ng/ml) for 3 days and S phase assessed by propidium iodide staining, as shown in panel (a). This data is from one donor, representative of three donors tested. (d) Reovirus treatment blocks IL‐15 induced expression of cell cycle mediators. PBMC were cultured for 4 h alone or primed with 1 MOI reovirus (Reo). 10 ng/ml IL‐15 was then added directly to all samples for 3 days. Total NK cells were isolated and MCM4, cyclin B and CDK2 analysed by immunoblotting along with β‐actin as a loading control. The blot image has been cut between the unstimulated control and cytokine treated lanes as shown by the boxing. These data are representative of three donors tested. (e) Modulation of IL‐15 mediated signalling by reovirus treatment. PBMC were cultured for 48 h alone (no virus) or primed with 1 MOI reovirus (+reovirus). After 48 h, 0, 1 or 10 ng/ml IL‐15 was added (as indicated) for 30 min. Phospho‐STAT1, STAT3, STAT5, mTOR and Akt were analysed by intracellular staining and flow cytometry, gating on the NK cell population. Graphs show median fluorescence intensities (MFI) for two or three separate donors, with standard deviation. Data were analysed by two‐way repeated‐measures ANOVA. When the effect of the virus was statistically significant, a post hoc Sidak multiple comparison tests was applied to identify statistically significant differences between “no virus” and “reovirus” MFI values; * p < 0·05

Journal: Immunology

Article Title: Oncolytic virus treatment differentially affects the CD56 dim and CD56 bright NK cell subsets in vivo and regulates a spectrum of human NK cell activity

doi: 10.1111/imm.13453

Figure Lengend Snippet: Reovirus‐induced IFN‐I production inhibits IL‐15‐mediated proliferation of NK cells. (a) Summary of the approach; Conditioned media (CM) was collected from reovirus treated (or untreated) PBMC and filtered to remove virions. This CM was applied to fresh PBMC (labelled with CFSE) together with IL‐15, in the presence or absence of IFN‐I blocking antibodies. PBMC were cultured for 5 days and proliferation of CD56 bright NK cells analysed by CFSE content. (b) Inhibition of CD56 bright NK cell proliferation by reovirus‐induced IFN‐I. CFSE analysis of CD56 brigh t NK cells following treatment described in (a). The percentage of cells that have proliferated after 5 days are indicated. These data are from a single donor, representative of three donors. (c) IFN‐I blocks IL‐15 induced S phase. Purified NK cells were cultured with IL‐15 or IL‐15+IFN‐I (all at 100 ng/ml) for 3 days and S phase assessed by propidium iodide staining, as shown in panel (a). This data is from one donor, representative of three donors tested. (d) Reovirus treatment blocks IL‐15 induced expression of cell cycle mediators. PBMC were cultured for 4 h alone or primed with 1 MOI reovirus (Reo). 10 ng/ml IL‐15 was then added directly to all samples for 3 days. Total NK cells were isolated and MCM4, cyclin B and CDK2 analysed by immunoblotting along with β‐actin as a loading control. The blot image has been cut between the unstimulated control and cytokine treated lanes as shown by the boxing. These data are representative of three donors tested. (e) Modulation of IL‐15 mediated signalling by reovirus treatment. PBMC were cultured for 48 h alone (no virus) or primed with 1 MOI reovirus (+reovirus). After 48 h, 0, 1 or 10 ng/ml IL‐15 was added (as indicated) for 30 min. Phospho‐STAT1, STAT3, STAT5, mTOR and Akt were analysed by intracellular staining and flow cytometry, gating on the NK cell population. Graphs show median fluorescence intensities (MFI) for two or three separate donors, with standard deviation. Data were analysed by two‐way repeated‐measures ANOVA. When the effect of the virus was statistically significant, a post hoc Sidak multiple comparison tests was applied to identify statistically significant differences between “no virus” and “reovirus” MFI values; * p < 0·05

Article Snippet: For IFN‐I ELISA, 96 well plates were coated with a mixture of antibodies against IFN‐α (Mabtech, MT1/3/5) overnight at 4°C, and blocked with PBS supplemented with 10% FCS.

Techniques: Blocking Assay, Cell Culture, Inhibition, Purification, Staining, Expressing, Isolation, Western Blot, Control, Virus, Flow Cytometry, Fluorescence, Standard Deviation, Comparison