human col1 α1  (Elabscience Biotechnology)

Journal: Science Advances

Article Title: Bacteriocin-transport–inspired oral peptide-probiotic delivery ameliorates IBD complications via autophagy and gut homeostasis

doi: 10.1126/sciadv.adz9069

Figure Lengend Snippet: ( A to D ) Antifibrosis efficacy of LL37 by quantitative polymerase chain reaction (qPCR) analysis of fibrotic markers (α-SMA, Col1α1, and FN) in TGF-β–stimulated CCD-18Co cells treated with LL37-1 (1.11 μM) or LL37-2 (11.1 μM). ( E ) Western blot assays of Col1α1 and FN protein expression. AU, arbitrary units; NC, negative control. ( F to H ) Cytotoxicity assessment of LL37 in (F) HT29 cells, (G) unstimulated CCD-18Co cells, and (H) TGF-β–pretreated (24 hours) CCD-18Co cells. ( I and J ) Cellular uptake of Cy5.5-labeled LL37 (20 μg ml −1 ) in unstimulated versus TGF-β–stimulated intestinal fibroblasts (I), with fluorescence intensity quantification (J). Scale bars, 100 μm. ( K ) Chromatogram profiles of LL37 detected by high-performance liquid chromatography (HPLC) following different treatments. ( L ) Fabrication schematic of BTB-Alg. ( M ) Zeta potential of Lac, LL37, and Alg. ( N ) Growth kinetics of Lac with LL37 coculture. ( O ) Fluorescence imaging of LL37 loading optimization [37.5, 75, 150, 300, 600, 1000, 1500, and 2500 μg of LL37 per 1 × 10 9 colony-forming units (CFU) of Lac]. ( P ) Fluorescence intensity of different BTB models. ( Q ) Zeta potential stabilization at saturation (Lac/LL 6 versus Lac/LL 7 ). n.s., not significant. ( R ) HPLC quantification of unbound LL37 in supernatants. ( S ) Alg coating optimization using Cy5.5-labeled Alg (0.032, 0.063, 0.125, 0.25, 0.5, 1, or 2 mg per 1 × 10 9 CFU of Lac). ( T ) Fluorescence intensity of Alg deposition. ( U and V ) Protective efficacy of Alg against pepsin degradation. HPLC chromatograms (U) and residual ratio of LL37 quantification (V). In (B) to (D), (F) to (H), (J), (M), (N), (P), (Q), (T), and (V), data are shown as mean ± SD ( n = 3 biological replicates). Statistical analysis was performed via a one-way analysis of variance (ANOVA) or an unpaired Student’s two-sided t test. Experiments in (E), (I), (K), (O), (R), (S), and (U) repeated three times with consistent results.

Article Snippet: Antibodies used for Western blot are listed: Col1α1 [Cell Signaling Technology (CST), #91144], FN (Proteintech, #15613-1-AP), and GAPDH (CST, #5174).

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Expressing, Negative Control, Labeling, Fluorescence, High Performance Liquid Chromatography, Zeta Potential Analyzer, Imaging

Journal: Science Advances

Article Title: Bacteriocin-transport–inspired oral peptide-probiotic delivery ameliorates IBD complications via autophagy and gut homeostasis

doi: 10.1126/sciadv.adz9069

Figure Lengend Snippet: ( A ) Experimental design of DSS-induced IF model and the timeline of therapeutic intervention. ( B ) Body weight changes during whole treatment. ( C and D ) Colon morphology (C) and length quantification (D) in different groups. ( E ) Histopathological analysis via H&E, Masson’s trichrome, and Van Gieson (VG) staining. Scale bars, 500 μm. ( F and G ) Quantitative histological scoring of inflammation (F) and fibrosis severity (G). ( H and I ) Immunofluorescence imaging of Col I (H) and α-SMA (I). Scale bars, 200 μm. ( J to L ) qPCR analysis of fibrotic markers α-SMA (J), Col1α1 (K), and FN (L) in CCD-18Co cells (C LL37 = 1.11 μM). ( M ) EMT modulation with vimentin (green) and E-cadherin (red) costaining. Scale bars, 100 μm. In (B), (D), (F), and (G), data are shown as mean ± SD ( n = 6 biological replicates). In (J) to (L), data are shown as mean ± SD ( n = 3 biological replicates). Statistical analysis was studied via one-way ANOVA, with P values denoting the statistical significance among the IF model, LL37, Lac, and Lac/LL@Alg groups. The images in (E), (H), (I), and (M) are representative of five biological replicates.

Article Snippet: Antibodies used for Western blot are listed: Col1α1 [Cell Signaling Technology (CST), #91144], FN (Proteintech, #15613-1-AP), and GAPDH (CST, #5174).

Techniques: Staining, Immunofluorescence, Imaging

Journal: Science Advances

Article Title: Bacteriocin-transport–inspired oral peptide-probiotic delivery ameliorates IBD complications via autophagy and gut homeostasis

doi: 10.1126/sciadv.adz9069

Figure Lengend Snippet: ( A ) Workflow for antifibrotic mechanism investigation. ( B ) AMPK activation levels in intestinal stricture tissues versus normal tissues from patients with CD. ( C ) Western blot analysis of AMPK activation and fibrotic marker gene expression after treatment with LL37 or Lac/LL@Alg (C LL37 = 20 μg ml −1 ). ( D and E ) Grayscale analysis of Western blot bands for phospho-AMPK (pAMPK)/AMPK (D) and FN (E). ( F to H ) qPCR level of α-SMA (F), Col1α1 (G), and FN (H) in CCD-18Co cells after different treatments. ( I ) Expression levels of autophagy-related genes between patients with active-stage CD and healthy controls in the GSE75214 dataset. ( J ) Dysregulation of autophagy-related genes in structured intestinal tissues compared to nonstructured tissues from patients with CD ( n = 9). ( K and L ) Autophagic flux dynamics were quantified through LC3B-II/I ratio modulation, p62 accumulation (K), and phospho-mTOR signaling activity (L). ( M ) GSEA of G protein signaling pathways and apelin signaling pathway activation in colon tissues after Lac/LL@Alg treatment. ( N ) Dynamic snapshot of the interaction between LL37 and apelin receptor (APJ). ( O ) Root mean square fluctuation (RMSF) analysis of the LL37-APJ and apelin-36–APJ complexes. ( P ) Average binding free energies of the simulated LL37-APJ or apelin-APJ complexes. In (B), (C), (K), (L), and (M) to (P), experiments were repeated three times with consistent results. In (D) to (H), data are shown as mean ± SD ( n = 3 biological replicates). Statistical analysis was studied via one-way ANOVA comparing phosphate-buffered saline (PBS), Met, LL37, LL37 + CC, Lac/LL@Alg, and Lac/LL@Alg + CC.

Article Snippet: Antibodies used for Western blot are listed: Col1α1 [Cell Signaling Technology (CST), #91144], FN (Proteintech, #15613-1-AP), and GAPDH (CST, #5174).

Techniques: Activation Assay, Western Blot, Marker, Gene Expression, Expressing, Activity Assay, Protein-Protein interactions, Binding Assay, Saline