Journal: PLoS ONE

Article Title: Pro-Inflammatory Action of MIF in Acute Myocardial Infarction via Activation of Peripheral Blood Mononuclear Cells

doi: 10.1371/journal.pone.0076206

Figure Lengend Snippet: A . Representative images showing a temporal changes of macrophages (CD68+ cells, purple colour) by immunohistochemical staining in mice treated with isotype control IgG or anti-MIF polyclonal antibody (MIF Ab) post MI. Bar=100 μm. B , C . Grouped data showing that neutralizing MIF by anti-MIF Ab, i.p. given immediately after MI, significantly reduced density of macrophages ( B , CD68 positive cells) and leukocytes ( C , CD45+ cells) at 24 h, but had no effect on macrophage density at 7 days. n=3 for sham-operated groups (SH) and n=6-7 for each time point of MI groups. * P<0 . 05 vs. respective SH, † P<0.05 vs. respective 24 h MI, # P <0.05. D . Representative immunoblotting images for monocyte chemoattractant protein 1 (MCP-1) and CD74 in hearts from sham-operated or MI (24 h) mice treated with isotype control IgG or MIF Ab. E , F . Quantitative analysis of MCP-1 ( E ) and CD74 ( F ) expression. n=3/per sham group, n=5/per MI group. * P<0 . 05 vs. sham, # P<0.05 . G . Effects of anti-MIF Ab and isotype control IgG (CTL) treatment on healing parameters, i.e. size of residual necrotic myocardium and collagen content in the infarct area, and infarct wall thickness at 7 days post MI. n=4-6/group. H . Representative photos of ruptured hearts that occurred at 3-4 days after MI. Arrows indicate the rupture site around the border zone. I . Treatment with the MIF antagonist, COR100140, in the first 3 days post MI significantly reduced incidence of cardiac rupture. n=25 for untreated and 15 for treated groups.

Article Snippet: To investigate the effect of anti-MIF intervention on inflammatory responses, animals were treated with a single dose of anti-MIF polyclonal antibody (Santa Cruz, sc-20121) or isotype control rabbit IgG (Santa Cruz, sc-2027), at 5 mg/kg i.p. immediately after CAO.

Techniques: Immunohistochemical staining, Staining, Control, Western Blot, Expressing

Journal: PLOS Pathogens

Article Title: DCLK1 isoform (DCLK1-S) as a critical player in promoting inflammation, tissue remodeling, and EMT in mouse models of colitis

doi: 10.1371/journal.ppat.1013360

Figure Lengend Snippet: A. Paraffin sections prepared from the colons of Control, CR or CR+DBZ group of Dclk1 ΔIEC mice were subjected to IMC. MMP13 (green) is overlayed with DCLK1 (red) and DNA (blue). Boxed areas in CR group indicates significant co-localization of MMP13 with DCLK1. Scale bars as indicated (100 μ m); 8-10 mice /group. B. Box plots of DCLK1 and MMP13 counts based on IMC data set in the Control, CR, CR+DBZ groups. C. DCLK1 and MMP13 staining from IMC in control and Dclk1-S OE group after DSS-induced colitis. Lane 1 DCLK1(red) staining is overlayed with DNA (blue), Lane 2 MMP13 (red) staining is overlayed with DNA (blue), Lane 3 DCLK1(red)/MMP13(green)/DNA(Blue) colocalization in the Dclk1-S OE mice. P values as indicated. D. MMP13 promoter-reporter activity (*, ** p <0.05; n = 3 independent experiments). Ei. Western blot data of HCT116 colon cancer cells treated with PMA and PMA+DBZ. Eii. In silico molecular docking studies to predict MMP13 and DCLK1 binding. CR: Citrobacter rodentium , CR+DBZ: Citrobacter rodentium + Dibenzazepine (DBZ), PMA: Phorbol 12-Myristate 13-Acetate.

Article Snippet: By utilizing RKO colon cancer cell line, we discovered that in response to PMA, a significant increase in MMP13 enzymatic activity was recorded with a dose-dependent decrease in response to MMP13 inhibitor WAY-170523 (Cat.# 2633, TOCRIS Biosciences; ).

Techniques: Control, Staining, Activity Assay, Western Blot, In Silico, Binding Assay

Journal: PLOS Pathogens

Article Title: DCLK1 isoform (DCLK1-S) as a critical player in promoting inflammation, tissue remodeling, and EMT in mouse models of colitis

doi: 10.1371/journal.ppat.1013360

Figure Lengend Snippet: RKO cells were treated with PMA or PMA plus selective and potent MMP13 inhibitor WAY 170523 at varying doses as indicated for 30 min followed by measurement of enzymatic activity. A. Reference curve showing relative fluorescence units (RFUs). B, C. Dose-dependent decrease in MMP13 enzymatic activity (n = 3 independent experiments; * p <0.05). D. Western blots showing relative protein abundance in three colon cancer cell lines. Boxed area represents levels of the indicated proteins in RKO cells. E. Subcellular compartmentalization of proteins from RKO cells. 1: Cytosolic Fraction, 2: Nuclear Fraction, 3: Membrane Fraction, 4: Cytoskeletal fraction (n = 3 independent experiments). F. Promoters for DCLK1-L and DCLK1-S isoforms were cloned and transfected in HEK293 cells and promoter-reporter activity assays were performed using Dual-Luciferase Reporter Assay System (E1910, Promega, Madison, WI). Luminescence was measured using a BioTek Synergy Neo luminometer. P values as indicated; n = 3 independent experiments.

Article Snippet: By utilizing RKO colon cancer cell line, we discovered that in response to PMA, a significant increase in MMP13 enzymatic activity was recorded with a dose-dependent decrease in response to MMP13 inhibitor WAY-170523 (Cat.# 2633, TOCRIS Biosciences; ).

Techniques: Activity Assay, Fluorescence, Western Blot, Quantitative Proteomics, Membrane, Clone Assay, Transfection, Luciferase, Reporter Assay

Journal: PLOS Pathogens

Article Title: DCLK1 isoform (DCLK1-S) as a critical player in promoting inflammation, tissue remodeling, and EMT in mouse models of colitis

doi: 10.1371/journal.ppat.1013360

Figure Lengend Snippet: A-D. Molecular docking study showing the interaction of proMMP13 (A) and mature MMP13 (C) with DCLK1-S. B. Ser 73 and Ser 114 highlighted in green in proMMP13, represent the phosphorylation sites for DCLK1-S. D. Arg207 and Gln211 are donor sites from MMP13 in mature protein close to Thr197 (circled). E. Both rhMMP13 and rhDCLK1 were incubated in kinase buffer in presence or absence of ATP at 30 °C for 1 hr. rhMMP13 didn’t show any autophosphorylation in the same condition. Phosphorylation was detected by pan phospho-Serine/Threonine antibody. F. rhMMP13 was incubated with rhDCLK1 in presence of ATP at 30 °C for 1 hr. Phosphorylated rhMMP13 was detected with immunoblotting using pan phospho-Serine/Threonine antibody. Representative immunoblots showing the levels of phosphorylation of rhMMP13. Right panel represents the Coomassie staining (n = 3 independent experiments). G, H. Protein band intensity ratios, pMMP13/pDCLK1 and pMMP13/Total MMP13, showing rhMMP13 phosphorylation by rhDCLK1.

Article Snippet: By utilizing RKO colon cancer cell line, we discovered that in response to PMA, a significant increase in MMP13 enzymatic activity was recorded with a dose-dependent decrease in response to MMP13 inhibitor WAY-170523 (Cat.# 2633, TOCRIS Biosciences; ).

Techniques: Phospho-proteomics, Incubation, Western Blot, Staining

Journal: PLOS Pathogens

Article Title: DCLK1 isoform (DCLK1-S) as a critical player in promoting inflammation, tissue remodeling, and EMT in mouse models of colitis

doi: 10.1371/journal.ppat.1013360

Figure Lengend Snippet: A. Tissue sections prepared from the colons of indicated groups of mice were subjected to immunostaining with antibodies against MMP13 (red), α -SMA (green), Collagen (green), E-cadherin (green) and Vimentin (green). Samples were analyzed using the Hyperion Imaging System (Standard BioTools). DAPI (blue) was used to label DNA. Scale bars = 100 μ m (n = 8-10mice/group). B. Box plots of MMP13 and Collagen counts based on IMC data set in the Control, CR and CR+DBZ groups. C. An overlay of Collagen (Red) and MMP13 (green) in tissue sections of the indicated groups. The boxed area represents a magnified image of collagen accumulation in the CR+DBZ group. Scale bars = 100 μ m (n = 8-10mice/group). D. t-SNE plots showing MMP13 intensity across different groups. E. Masson’s Trichrome staining of tissue sections prepared from the colons of Control, CR, and CR+DBZ mice. Please note increases in collagenous fibrous tissue (stained blue) in both CR and CR+DBZ groups. Scale bars = 200 μ m; n = 8-10mice/group. CR: Citrobacter rodentium , CR+DBZ: Citrobacter rodentium + Dibenzazepine (DBZ).

Article Snippet: By utilizing RKO colon cancer cell line, we discovered that in response to PMA, a significant increase in MMP13 enzymatic activity was recorded with a dose-dependent decrease in response to MMP13 inhibitor WAY-170523 (Cat.# 2633, TOCRIS Biosciences; ).

Techniques: Immunostaining, Imaging, Control, Staining