timp3 mouse  (Bio-Techne corporation)

Journal: The Journal of Immunology Author Choice

Article Title: The Metalloproteinase ADAMTS5 Is Expressed by Interstitial Inflammatory Cells in IgA Nephropathy and Is Proteolytically Active on the Kidney Matrix

doi: 10.4049/jimmunol.2000448

Figure Lengend Snippet: Identification of matrix proteins by urine proteomics leads to the discovery of ADAMTS5 in IgAN kidneys. ( A ) LC-MS/MS proteomics of healthy donor and IgAN patient urine identified multiple ECM proteins present in IgAN urine (see Ref. ). Matrix proteins were selected using gene ontology (BinGO) and are presented as a protein-protein interaction network. Yellow and red proteins are increased in IgAN urine, gray are unchanged, and blue are decreased in comparison with healthy samples. Proteomics analysis including all identified proteins is described in detail in . ( B ) The mRNA expression of ADAMTS5 and TIMP3 was examined in a published microarray dataset comparing 25 IgAN patients with six healthy donor kidney biopsies. Microarray gene expression was retrieved directly from National Center for Biotechnology Information–Gene Expression Omnibus (GSE35488; https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE35488 ). Microarray data are available in . ADAMTS5 and TIMP3 were significantly regulated in IgAN . Differences were examined using standard t test using data curated and processed by NCBI-GEO as described in GSE35488; https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE35488 . The p values are shown. ( C ) ADAMTS5 protein immunofluorescence was measured in 13 healthy (kidney transplant donors) versus 20 IgAN patients. IgAN donors were biopsy diagnosed. ADAMTS5 staining was quantified using ADAMTS5-stained particle counting corrected to biopsy tissue area on ImageJ and values were compared using two-tailed t test. ( D ) For the 20 IgAN patients, ADAMTS5 immunofluorescence was correlated to their matching urine protein concentration (proteinuria; mg/dl) and eGFR (estimated glomerular filtration rate; ml/min/1.73 m 2 ) values. The r and p values were computed using nonparametric Spearman test given the dissimilar nature of the correlated values. ( E ) ADAMTS5 staining was also compared in the same IgAN biopsies but biopsies were grouped according to their histological MEST-C score. We focused on the (T) score (percentage of biopsy area affected by tubular atrophy or interstitial fibrosis, whichever is greater) and compared less affected T0 biopsies ( n = 7) with more affected T1/T2 biopsies ( n = 13). There was a significant increase in ADAMTS5 staining in T1/T2 specimens ( t test). ( F ) ADAMTS5 biopsy staining was also measured separately in the tubulointerstitium and glomeruli and compared in healthy versus IgAN biopsies. ADAMTS5 staining in the IgAN interstitium ( IgAN Interst ) is significantly increased in comparison with staining in IgAN glomeruli ( IgAN Glom ), as well as in comparison with healthy interstitium and glomeruli, which are NS different to each other (ANOVA with Fisher least significant difference, multiple comparison test). ( G and H ) Examples of ADAMTS5 immunofluorescence in IgAN and healthy kidney biopsies costained with relevant proteins. In (G) ADAMTS5 (red) was costained with ADAMTS1 (green) and TIMP3 (blue). TIMP3 staining was below the threshold of confident detection in either IgAN or healthy biopsies. In (H) ADAMTS5 (red) was costained with TIMP1 (green). ADAMTS5 is close but does not appear to colocalise with TIMP1 in affected tubulointerstitial areas. tub denotes renal tubules and glom denotes glomeruli. Note that ADAMTS5 is increased in areas of tubulointerstitial inflammatory infiltration and remodelling. IgAN glomeruli also contain ADAMTS5 + cells. In healthy biopsies, there were sporadic ADAMTS5-stained (red) cells in the interstitium [(G) healthy] and glomeruli [(H) healthy]. White-dashed boxes denote areas shown in higher magnification in lower panels.

Article Snippet: Sections were treated using acid-based Ag retrieval and dewaxing solution (Abcam) at 98°C for 1 h. Sections were thoroughly washed, blocked in 10% normal donkey serum (Dako) for 1 h, and subsequently incubated with anti-human primary Abs against the following proteins: ADAMTS5 (raised in rabbit), CD64, and vimentin (mouse) from Abcam and TIMP3 (mouse), myeloperoxidase, TIMP1, lumican (LUM), ADAMTS1, versican (VCAN), collagen-4, and CD206 (all goat) from Bio-Techne.

Techniques: Liquid Chromatography with Mass Spectroscopy, Comparison, Expressing, Microarray, Immunofluorescence, Staining, Two Tailed Test, Protein Concentration, Filtration

Journal: bioRxiv

Article Title: Regulatory mechanisms in multiple vascular diseases locus LRP1 involve repression by SNAIL and extracellular matrix remodeling

doi: 10.1101/2023.05.09.539992

Figure Lengend Snippet: A : Illustration of indel frameshift in exons of LRP1 to generate knockout iPSCs using CRISPR/Cas9 prior to differentiation into SMCs B: Representative Western blot images showing LRP1 protein expression in WT and LRP1 KO iPSC-SMCs. C : Heatmap representation of relative RNA expression for differentially expressed genes between LRP1 KO and WT iPSC-SMCs. Genes involved in “collagen containing extracellular matrix” (gene ontology 0062023) are indicated D : Representative Western blot images showing the expression of phospho-SMAD2/3 (p-SMAD2/3) and total SMAD2/3 (SMAD2/3) in WT and LRP1 KO iPSC-SMCs in the presence of TGFβ1 protein for 1 hour. E : Heatmap representation of label free quantification (LFQ) for proteins over or underrepresented in extracellular extracts of LRP1 KO and WT iPSC-SMCs. F : Representative Western blot images showing the expression of LRP1, CYR61 and TIMP3 in whole cell extracts or decellularized extracts (ECM).

Article Snippet: Following antibodies were used in Western blot experiments: rabbit anti-LRP1 (ab92544, Abcam, Cambridge, UK), mouse anti-β-actin (sc-47778, Santa Cruz Biotechnology, Dallas, Texas, USA), rabbit anti-phospho-SMAD2/3 (#8828) and anti-SMAD2/3 (#3102, Cell Signaling Technology, Danvers, Massachusetts, USA), mouse anti-TIMP3 (LS-B2576, Lifespan Biosciences, Seattle, Washington, USA), mouse anti-CYR61 (sc-374129, Santa Cruz Biotechnology, Dallas, Texas, USA).

Techniques: Knock-Out, CRISPR, Western Blot, Expressing, RNA Expression

Journal: Advanced healthcare materials

Article Title: In Situ Deployment of Engineered Extracellular Vesicles into the Tumor Niche via Myeloid-Derived Suppressor Cells.

doi: 10.1002/adhm.202101619

Figure Lengend Snippet: Fig. 1. Non-viral transfection of MDSCs mediates the release of engineered EVs with desirable cargo. (a) Schematic diagram illustrating the experimental design. (1) MDSCs were electroporated with expression plasmids for Timp3 or Rarres2. Electroporation with sham/empty plasmids served as control. (2) The plasmids are expressed within the MDSCs and (3) transcripts are packed and released within EVs. qRT-PCR analysis of the MDSC cultures at (b) 12, (c) 24, (d) 48, and (e) 72 hours post-electroporation reveals strong Timp3 or Rarres2 overexpression. Analysis of the EVs isolated from the supernatant at (f) 24 and (g) 48 hours

Article Snippet: Plasmid vector Company Cat. No Backbone Sham Origene PS100001 pCMV6 Timp3 (Mouse tissue inhibitor metalloproteinase 3) Origene MG202295 pCMV6 Rarres2 (Mouse retinoic acid receptor responder) Origene MG222586 pCMV6 Adv Healthc Mater.

Techniques: Transfection, Expressing, Electroporation, Control, Quantitative RT-PCR, Over Expression, Isolation

Journal: Advanced healthcare materials

Article Title: In Situ Deployment of Engineered Extracellular Vesicles into the Tumor Niche via Myeloid-Derived Suppressor Cells.

doi: 10.1002/adhm.202101619

Figure Lengend Snippet: Fig. 2. MDSC-derived EVs can be internalized by cancer cells and modulate gene expression. (a) Schematic diagram illustrating the experimental design. MDSCs were electroporated with expression plasmids for Timp3 or Rarres2. Electroporation with sham/empty plasmids served as control. MDSC-derived EVs were isolated and incubated with Py8119 mouse breast cancer cell cultures for 6 – 24 hours. (b) Fluorescence microscopy imaging of the Py8119 cells (labeled red and blue) revealed successful uptake of MDSC-derived EVs (labeled green). The images shown represent EV uptake at 24 h post-exposure. qRT-PCR analysis of the Py8119 cultures at (c) 6, (d) 12, and (d) 24 hours post-EV exposure reveals strong Timp3 or Rarres2 overexpression after 12 hours of exposure. ** p< 0.01 (n= 4), **** p< 0.0001 (n= 4).

Article Snippet: Plasmid vector Company Cat. No Backbone Sham Origene PS100001 pCMV6 Timp3 (Mouse tissue inhibitor metalloproteinase 3) Origene MG202295 pCMV6 Rarres2 (Mouse retinoic acid receptor responder) Origene MG222586 pCMV6 Adv Healthc Mater.

Techniques: Derivative Assay, Gene Expression, Expressing, Electroporation, Control, Isolation, Incubation, Fluorescence, Microscopy, Imaging, Labeling, Quantitative RT-PCR, Over Expression

Journal: Advanced healthcare materials

Article Title: In Situ Deployment of Engineered Extracellular Vesicles into the Tumor Niche via Myeloid-Derived Suppressor Cells.

doi: 10.1002/adhm.202101619

Figure Lengend Snippet: Fig. 3. Transfected MDSCs can transfer engineered EVs to breast cancer cells, in situ, and mediate gene expression and cellular responses. (a) Schematic diagram illustrating the experimental design. MDSCs were electroporated with expression plasmids for Timp3 or Rarres2. Electroporation with sham/empty plasmids served as control. Transfected MDSCs and Py8119 mouse breast cancer cells were co- cultured using a transwell system, with the MDSCs in the apical chamber and Py8119 cells in the basal chamber. The MDSCs were-prelabeled with a membrane dye to trace EV release and uptake. MDSC-derived EVs were thus expected to translocate across the membrane

Article Snippet: Plasmid vector Company Cat. No Backbone Sham Origene PS100001 pCMV6 Timp3 (Mouse tissue inhibitor metalloproteinase 3) Origene MG202295 pCMV6 Rarres2 (Mouse retinoic acid receptor responder) Origene MG222586 pCMV6 Adv Healthc Mater.

Techniques: Transfection, In Situ, Gene Expression, Expressing, Electroporation, Control, Cell Culture, Membrane, Derivative Assay

Journal: Advanced healthcare materials

Article Title: In Situ Deployment of Engineered Extracellular Vesicles into the Tumor Niche via Myeloid-Derived Suppressor Cells.

doi: 10.1002/adhm.202101619

Figure Lengend Snippet: Fig. 4. Transfected MDSCs can also modulate cancer cell proliferation and invasion, in situ. (a) Schematic diagram illustrating the experimental design. MDSCs were electroporated with expression plasmids for Timp3 or Rarres2. Electroporation with sham/empty plasmids served as control. Transfected MDSCs and Py8119 mouse breast cancer cells were co- cultured in direct contact. Matrigel-coated transwell insets were used for cell invasion studies. The MDSCs were-prelabeled green, and the Py8119 cells were prelabeled red. (b) Fluorescence microscopy imaging of the MDSC/Py8119 co-cultures after 24 hours. (c) Flow cytometry analysis revealed reduced Py8119 cell numbers in co-cultured with Timp3- or

Article Snippet: Plasmid vector Company Cat. No Backbone Sham Origene PS100001 pCMV6 Timp3 (Mouse tissue inhibitor metalloproteinase 3) Origene MG202295 pCMV6 Rarres2 (Mouse retinoic acid receptor responder) Origene MG222586 pCMV6 Adv Healthc Mater.

Techniques: Transfection, In Situ, Expressing, Electroporation, Control, Cell Culture, Fluorescence, Microscopy, Imaging, Flow Cytometry

Journal: Advanced healthcare materials

Article Title: In Situ Deployment of Engineered Extracellular Vesicles into the Tumor Niche via Myeloid-Derived Suppressor Cells.

doi: 10.1002/adhm.202101619

Figure Lengend Snippet: Fig. 5. Transfected MDSCs retain tumor-homing abilities and drive anti-tumoral gene and protein expression. (a) Schematic diagram illustrating the experimental design. MDSCs were electroporated with expression plasmids for Timp3 or Rarres2. Electroporation with sham/empty plasmids served as control. Transfected MDSCs were then injected into tumor-bearing mice via the tail vein, and accumulation and gene/protein expression in the tumor was evaluated after 24 hours. (b, c) IVIS imaging after 24 hours revealed strong accumulation of transfected MDSCs in the tumor niche compared to other organs. Transfected MDSCs were

Article Snippet: Plasmid vector Company Cat. No Backbone Sham Origene PS100001 pCMV6 Timp3 (Mouse tissue inhibitor metalloproteinase 3) Origene MG202295 pCMV6 Rarres2 (Mouse retinoic acid receptor responder) Origene MG222586 pCMV6 Adv Healthc Mater.

Techniques: Transfection, Expressing, Electroporation, Control, Injection, Imaging