Journal: Scientific reports

Article Title: FimH-based display of functional eukaryotic proteins on bacteria surfaces.

doi: 10.1038/s41598-019-44883-z

Figure Lengend Snippet: Figure 2. Design and expression of POI-FimH fusion proteins. (A) The Gaussia Luciferase (GLuc) is N-linked to the FimH protein and thereby becomes part of the fimbriae on the bacterial surface. By use of the bacterial pet11d vector, the expression of the GLuc-FimH fusion protein is induced by incubation with IPTG. (B) Schematic representation of the expression cassettes for the FimH fusion proteins with human EGF, human TGF-α and human epiregulin EPRG, respectively. Gly, Glycine-(Gly-Gly-Gly-Gly-Ser)3-linker; cMyc, c-Myc tag; CS, HRV3C protease cleavage site. (C) The expression of the POI-FimH protein on the surface of E. coli was induced by incubation with IPTG for 2 hrs and recorded by flow cytometry. Bacteria containing the GLuc- FimH expression vector were stained with a Gaussia luciferase-specific rabbit antibody and detected with a PE- coupled anti-rabbit antibody (bold histogram). An isotype-matched PE-conjugated antibody (light histogram) and non-modified E. coli (wt) served as controls. The expression of EGF-FimH, TGFα-FimH and Epiregulin- FimH on the surface of engineered E. coli bacteria was induced by IPTG. The bacteria were stained with a PE labeled c-Myc-specific mouse antibody and the proteins were recorded by flow cytometry (bold histograms). An isotype-matched PE-conjugated antibody (light histograms) served as control. All experiments were performed in triplicates and a representative histogram is shown.

Article Snippet: The detection of the proteins of interest was performed by using ELISA kits specific for human EGF, TGF-α and EPRG, respectively (CUSABIO BIOTECH, Houston, TX, USA).

Techniques: Expressing, Luciferase, Plasmid Preparation, Incubation, Ubiquitin Proteomics, Flow Cytometry, Bacteria, Staining, Modification, Labeling, Control

Journal: Nature Communications

Article Title: Therapeutic potential of the secreted Kazal-type serine protease inhibitor SPINK4 in colitis

doi: 10.1038/s41467-024-50048-y

Figure Lengend Snippet: a Relative expression of IEC markers from different branches, including enteroendocrine cells ( Neurog3 , Insm , and Pax4 ), secretory lineage ( Muc2 , Gfi1 , Foxa2 , Reg3a , and Lyz ), M cells ( Spib ), Tuft cells ( Dclk ), stem cells ( Olfm4 , Lgr5 ), and absorptive cells ( Si ) in mouse organoids with rSPINK4 stimulation. The degree of color depth indicates the abundance of expression positively. b Levels of phosphorylated and total EGFR in NCM460 and HT-29 cells with 5 ng/mL rSPINK4 stimulation for 0–60 min. c Direct downstream molecules of the EGFR pathway detected using western blotting with rSPINK4 stimulation for 0–60 min. d Typical fluorescent images of intestinal organoids from WT and KO mice including MUC2 (green), E-cadherin (red), and DAPI (blue), with rSPINK4 (100 ng/mL) and AG1478 (10 μM) stimulation under inflammatory conditions (50 ng/mL TNF-α treatment). e Quantitative analysis of weight loss with SPINK4 and gefitinib intervention following DSS administration in the control group drinking water (control, n = 4); DSS and PBS administration group (DSS + PBS, n = 6); DSS and rSPINK4 administration group (DSS + rSPINK4, n = 5); and DSS, rSPINK4, and gefitinib administration group (DSS + rSPINK4 + gefitinib, n = 4). f Representative canonical endoscopic images with the lesion circled with white arrows. g Quantification of colonic length. h The levels of the phosphorylated and total EGFR were determined in WT and KO mouse tissues (upper panel) and rSPINK4 treatment group (lower panel). Data are presented as the mean ± SEM. All tests were two-sided. Statistical significance was calculated using unpaired Student’s t test ( h ). Besides, Kruskal–Wallis test ( h ) and one-way ANOVA ( b , e , g ) were performed for multiple comparison; n = 3–5 biologically independent experiments ( b , g , h ). Source data are provided as a Source Data file.

Article Snippet: The organoids generated from both human and mouse tissues were cultured in the medium without EGF, which synergistically affects the EGFR pathway, and supplemented with 100 ng/mL rSPINK4 and 10 μM AG1478 (MCE, USA) for 48 h. The inflammatory condition could be developed using 50 ng/mL TNF-α.

Techniques: Expressing, Western Blot, Control, Comparison

Journal: Nature Communications

Article Title: Therapeutic potential of the secreted Kazal-type serine protease inhibitor SPINK4 in colitis

doi: 10.1038/s41467-024-50048-y

Figure Lengend Snippet: a The FLAG-SPINK4 protein, which exists in the concentrated medium of SPINK4- overexpressing cells, is involved in the EGFR protein from cell lysis combined with EGFR antibody. IgG is the contrast to EGFR antibody. b The binding test of rSPINK4 protein and EGFR in membrane extraction under the incubation in vitro. c rSPINK4 coprecipitates with rEGFR (extracellular part) mutually in the in vitro pull-down assay controlled by IgG. d Representative immunofluorescent image of rSPINK4 and EGFR localization (EGFR: green, rSPINK4: red, DAPI: blue). e The curve of released heat and change in △H are performed when the rEGFR-His protein (6 μM) is titrated by rSPINK4-His protein (30 μM) via the ITC assay. f The pull-down assay of rEGF and rEGFR (extracellular part) in a SPINK4-dose-dependent manner. The concentration of rSPINK4 is shown. g A typical binding curve between biotinylated EGF and EGFR was performed using AlphaLISA binding assay. h Image profile of the interaction between rSPINK4 and the deleted extracellular domain of EGFR (△1: the deletion of 57–168 aa, △2: the deletion of 177–338 aa, △3: the deletion of 361–481 aa, △4: the deletion of 505–637 aa, ED: whole extracellular EGFR, NC: vector). i Interaction between mutant rSPINK4 (wt: whole sequence of SPINK4, mut1: mutation sites: C65A, C68A, and C86A, mut2: mutation sites: Q48A and M49A, NC: vector) and extracellular EGFR in vitro. j Predictive interactive model of SPINK4 and extracellular EGFR (pink: SPINK4, the tail is at N-terminal; red: EGFR subdomain including 57–168 aa; green: EGFR subdomain including 177–338 aa; yellow: EGFR subdomain including 361–481 aa; orange: EGFR subdomain including 505–637 aa). Data are presented as the mean ± SEM. The Statistical test was two-sided using one-way ANOVA ( g ); n = 6 biologically independent experiments ( g ). Source data are provided as a Source Data file.

Article Snippet: The organoids generated from both human and mouse tissues were cultured in the medium without EGF, which synergistically affects the EGFR pathway, and supplemented with 100 ng/mL rSPINK4 and 10 μM AG1478 (MCE, USA) for 48 h. The inflammatory condition could be developed using 50 ng/mL TNF-α.

Techniques: Lysis, Binding Assay, Membrane, Extraction, Incubation, In Vitro, Pull Down Assay, Isothermal Titration Calorimetry, Concentration Assay, Plasmid Preparation, Mutagenesis, Sequencing