human type iii collagen sirna  (Santa Cruz Biotechnology)

Journal: eLife

Article Title: Peptidoglycan-Chi3l1 interaction shapes gut microbiota in intestinal mucus layer

doi: 10.7554/eLife.92994

Figure Lengend Snippet: ( A ) Structural comparison between chitin and PGN. Both chitin and PGN contain N-acetylglucosamine (GlcNAc) and have β–1,4-glycosidic bonds in their structures. However, chitin is purely a polysaccharide, while PGN includes a peptide component that forms cross-links between chains . ( B ) Gram-positive bacteria ( E. faecalis , S. saprophyticus ) and Gram-negative bacteria ( E. coli ) were incubated with 1 μg of recombinant mouse Chi3l1 protein (rmChi3l1), respectively. Proteins bound to indicated bacteria were precipitated by centrifugation. Western blot was used to detect rmChi3l1 in Pellet, Supernatant (unbound proteins) and Last Wash (last wash unbound proteins). ( C ) Insoluble PGN were incubated with either recombinant mouse Chi3l1 protein (rmChi3l1) or bovine serum albumin (BSA). Proteins bound to PGN were precipitated by centrifugation. Silver staining was used to detect rmChi3l1 in Input, Supernatant (unbound proteins), Pellet and Last Wash (last wash unbound proteins). ( D ) Insoluble PGN were incubated with recombinant human Chi3l1 protein (rhChi3l1). Proteins bound to PGN were precipitated by centrifugation. Silver staining was used to detect rhChi3l1 in Input, Supernatant (unbound proteins), Pellet and Last Wash (last wash unbound proteins). All data above represent at least three independent experiments. ( E ) Insoluble PGN or chitin was incubated with rmChi3l1. Chi3l1 bound to PGN (upper panel) and chitin (lower panel) was precipitated and detected by silver staining. The supernatant represents the last wash, and the pellet contains proteins precipitated by either PGN or chitin. ( F ) Relative DLD-1 bacterial binding preference after treatment with K12 or GlmM, a PGN synthesis-deficient mutant. Colony-forming units (CFU) were counted, and GlmM CFU were normalized to 1. ( G ) Relative K12 bacterial adhesion preference after DLD-1 cells were transfected without (Mock), or with scramble shRNA (shCK), or with sh Chil1 . CFU were counted, and the Mock group were normalized to 1. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns: no significant difference, error bar indicates SEM. Figure 2—source data 1. File containing original western blots for and silver staining for , indicating the relevant bands. Figure 2—source data 2. Original files for western blot analysis displayed in and silver staining for . Figure 2—source data 3. Numerical data of .

Article Snippet: Recombinant DNA reagent , PLKO.1-Puro (plasmid) , Addgene , RRID: Addgene_10878 , Pol III-based shRNA backbone.

Techniques: Comparison, Bacteria, Incubation, Recombinant, Centrifugation, Western Blot, Silver Staining, Binding Assay, Mutagenesis, Transfection, shRNA

Journal: eLife

Article Title: Peptidoglycan-Chi3l1 interaction shapes gut microbiota in intestinal mucus layer

doi: 10.7554/eLife.92994

Figure Lengend Snippet:

Article Snippet: Recombinant DNA reagent , PLKO.1-Puro (plasmid) , Addgene , RRID: Addgene_10878 , Pol III-based shRNA backbone.

Techniques: Recombinant, Plasmid Preparation, shRNA, Transfection, Construct, Purification, Bacteria, Staining, Gel Extraction, SYBR Green Assay

Journal: NPJ Breast Cancer

Article Title: Prognostic and therapeutic implications of tumor-restrictive type III collagen in the breast cancer microenvironment

doi: 10.1038/s41523-024-00690-y

Figure Lengend Snippet: Human skin fibroblasts were transfected with non-targeting siRNA (siCtrl) or Col3 siRNA (siCol3). a , b After 96h, the efficiency and specificity of knockdown were confirmed by qPCR for COL3A1 and COL1A1 . c , d Representative Second Harmonic Generation (SHG) images of collagen within human fibroblast-derived matrices (FDMs) prepared from fibroblast/ECM units treated with control and Col3-targeted siRNA. Collagen matrices were analyzed for ( e ) total collagen amount and ( f ) collagen alignment. CT-FIRE was used to quantify ( g ) collagen fiber number, ( h ) straightness, ( i ) width, and ( j ) length from SHG images. Data from representative experiments are presented (5 individual experiments performed with similar results). Error bars show mean ± SD ( n = 5). Statistical significance was determined by unpaired Student t tests: * p < 0.05; ** p < 0.01; **** p < 0.0001. Scale bar = 25 µm.

Article Snippet: Human type III collagen siRNA, containing a pool of multiple Col3a1-specific siRNAs (sc-43062), and control siRNA (sc-37007) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Transfection, Knockdown, Derivative Assay, Control

Journal: NPJ Breast Cancer

Article Title: Prognostic and therapeutic implications of tumor-restrictive type III collagen in the breast cancer microenvironment

doi: 10.1038/s41523-024-00690-y

Figure Lengend Snippet: Human breast CAFs were transfected with non-targeting siRNA (siCtrl) or Col3 siRNA (siCol3). a , b After 5 days, the efficiency and specificity of knockdown were confirmed by qPCR for COL3A1 and COL1A1 . c , d Representative second harmonic generation (SHG) images of collagen within human CAF-derived matrices (FDMs) prepared from fibroblast/ECM units treated with control and Col3-targeted siRNA. SHG obtained images of collagen matrices were analyzed for ( e ) total collagen amount and ( f ) collagen alignment. Data represents individual FDMs from 3 independent experiments. g , h Representative images of FDMs prepared from breast CAFs cultured on with rhCol1- or rhCol3-coated substrata at 8 days. Collagen matrices were analyzed for ( i ) total collagen amount and ( j ) collagen alignment. Data represents individual FDMs from 2 independent experiments. Error bars show mean ± SD. Significance determined with Student’s unpaired t tests: * p < 0.05; *** p < 0.001; **** p < 0.0001. Scale bar = 25 µm.

Article Snippet: Human type III collagen siRNA, containing a pool of multiple Col3a1-specific siRNAs (sc-43062), and control siRNA (sc-37007) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Transfection, Knockdown, Derivative Assay, Control, Cell Culture