Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: RIPK3 protects against endothelial activation and vascular permeability in a mouse model of ischemia-reperfusion injury

doi: 10.1161/ATVBAHA.125.322977

Figure Lengend Snippet: ( A ) Schematic of mouse tissue collection and quantitative analysis of cytokines at 0 hr and 4 hr after I/R injury in Ripk3 WT and Ripk3 iECKO mice. ( B ) Baseline IL-6 levels (left) and fold change (from baseline) following 4 hr I/R injury (right) in small intestinal tissue of Ripk3 WT and Ripk3 iECKO mice (n=3–4/group). ( C ) Baseline IL-6 levels (left) and fold change (from baseline) following 4 hr I/R injury (right) in serum of Ripk3 WT and Ripk3 iECKO mice (n=3–4/group). ( D ) Schematic of IL-6 measurement in stimulated whole blood and MS1 endothelial cells (ECs). ( E and F ) Plasma IL-6 levels at 2 hr ( E ) and 4 hr ( F ) after ex-vivo stimulation of whole blood collected from Ripk3 WT and Ripk3 iECKO mice. At each time point, blood was stimulated with phosphate-buffered saline (PBS) (left) or lipopolysaccharide (LPS) (right); LPS data are graphed as fold change from PBS control treatments (n=3/group). ( G ) IL-6 levels in cell supernatants following stimulation of WT and Ripk3 -KO MS1 ECs with 20ng/mL TNFα for 0 hr and 24 hr (n=3/group). ( H ) Il6 transcripts measured by qRT-PCR in WT and Ripk3 -KO MS1 ECs following stimulation with 20ng/mL TNFα for 0 hr, 4 hr and 8 hr (n=4/group). Data represent mean ± SEM. P values were determined by two-tailed Student’s t -test ( B , C , E and F ) and two-way ANOVA ( G and H ).

Article Snippet: The MS1 adult murine pancreatic EC line (ATCC; #CRL 2279) was cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco; #11960–044) supplemented with 10% fetal bovine serum (FBS) (Fisher Scientific; 3SH30910.03) and 1% antibiotics (streptomycin/amphotericin; Sigma; #A5955).

Techniques: Clinical Proteomics, Ex Vivo, Saline, Control, Quantitative RT-PCR, Two Tailed Test

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: RIPK3 protects against endothelial activation and vascular permeability in a mouse model of ischemia-reperfusion injury

doi: 10.1161/ATVBAHA.125.322977

Figure Lengend Snippet: ( A ) Representative immunofluorescence images in grey scale depicting cytosolic versus nuclear NRF2 expression at baseline and 1 hr following 20ng/ml TNFα or tert-Butylhydroquinone (tBHQ) stimulation in WT and Ripk3 -KO MS1 ECs. Scale bar: 20 μm. ( B-D ) Mean NRF2 fluorescence intensity in cytosolic and nuclear compartments (left) and calculated NRF2 nuclear to cytosolic ratio (right) in WT and Ripk3 -KO MS1 ECs at baseline ( B ) or after 1 hr stimulation with TNFα ( C ) or tBHQ ( D ) (n=3/group). ( E-F ) Transcript expression following stimulation with 20ng/ml TNFα for 0 hr and 4 hr in WT and Ripk3 -KO MS1 ECs of the following genes: Hmox1 (heme oxygenase-1) ( E ) and Nqo1 [NAD(P)H quinone dehydrogenase 1] ( F ) (n=3–4/group). ( G ) Il6 transcript expression in Ripk3 -KO and Ripk3 -KO + NRF2 overexpression (OE) MS1 ECs following stimulation with 20ng/mL TNFα for 0 hr and 4 hr (n=3/group). ( H ) IL-6 levels in cell supernatants following stimulation of Ripk3 -KO and Ripk3 -KO+ NRF2 OE MS1 ECs with 20ng/mL TNFα for 0 hr, 4 hr and 24 hr (n=3/group). Data represent mean ± SEM. P values were determined by two-tailed Student’s t -test or two-way ANOVA ( B , C and D ) and two-way ANOVA ( E , F , G and H ).

Article Snippet: The MS1 adult murine pancreatic EC line (ATCC; #CRL 2279) was cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco; #11960–044) supplemented with 10% fetal bovine serum (FBS) (Fisher Scientific; 3SH30910.03) and 1% antibiotics (streptomycin/amphotericin; Sigma; #A5955).

Techniques: Over Expression, Immunofluorescence, Expressing, Fluorescence, Two Tailed Test

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: RIPK3 protects against endothelial activation and vascular permeability in a mouse model of ischemia-reperfusion injury

doi: 10.1161/ATVBAHA.125.322977

Figure Lengend Snippet: ( A ) VCAM-1 expression in the small intestine assessed by immunoblot in baseline Ripk3 WT and Ripk3 iECKO mice (n=7/group). ( B ) VCAM-1 expression assessed by immunoblot in WT and Ripk3 -KO MS1 endothelial cells (ECs) stimulated with 20ng/ml TNFα for 24 hr (n=4/group). ( C ) WT and Ripk3 -KO MS1 cells were stimulated with either PBS or 20ng/ml TNFα for 24 hr and immunostained for VCAM-1. White arrow heads in the insets indicate junctional localization of VCAM-1, Scale bar: 20 μm, inset = 10 μm. ( D ) VCAM-1 expression assessed by immunoblot in WT and Ripk3 -KO MS1 ECs stimulated with 20ng/ml TNFα for 24 hr in the presence of control IgG antibody or neutralizing IL-6 antibody (n=4/group). Data represent mean ± SEM. P values were determined by two-tailed Student’s t -test ( A ) and two-way ANOVA ( B and D ).

Article Snippet: The MS1 adult murine pancreatic EC line (ATCC; #CRL 2279) was cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco; #11960–044) supplemented with 10% fetal bovine serum (FBS) (Fisher Scientific; 3SH30910.03) and 1% antibiotics (streptomycin/amphotericin; Sigma; #A5955).

Techniques: In Vivo, In Vitro, Expressing, Western Blot, Control, Two Tailed Test

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: RIPK3 protects against endothelial activation and vascular permeability in a mouse model of ischemia-reperfusion injury

doi: 10.1161/ATVBAHA.125.322977

Figure Lengend Snippet: ( A ) CD45 + leukocyte accumulation was assessed by immunostaining of small intestinal tissues from Ripk3 WT and Ripk3 iECKO mice following 24 hr I/R injury. Scale bar: 100 μm. ( B ) CD45 + cells, as in A, were quantified from multiple confocal Z-stack images taken from each mouse tissue by normalizing CD45 + expression area to total cell nuclear area in small intestines. Individual data points are averages of 2–4 imaged and quantified sections per mouse (n=5/group). ( C ) Representative images of immortalized BMA3.1A7 macrophages labeled with calcein-AM fluorescent dye (green) and incubated on top of a monolayer of WT and Ripk3 -KO MS1 endothelial cells (ECs) that were previously treated with 20ng/mL TNFα for 24 hr. Cells were washed multiple times before imaging. Scale bar: 20 μm. (D) The number of macrophages adherent to ECs was quantified in each acquired image (n=4 images/group). ( E ) Following imaging (as in C), cells were lysed with Triton-X, and fluorescence was measured using a plate reader. Values were normalized to readings from wells in which BMA3.1A7 cells were incubated with untreated WT MS1 ECs (dotted line) (n=3/group). ( F ) Timeline for detecting vascular permeability with Evans blue dye (EBD) after clodronate-liposome treatment and intestinal I/R injury; tamoxifen was administered at 8 weeks of age. ( G ) Small intestinal vascular permeability (fold change from each genotype’s own baseline) after 24 hr intestinal I/R injury; measurements are shown in the injury region (I/R) and control regions (control, distal control) following clodronate-liposome treatment (n=4–5/group). ( H ) CD45 + leukocyte accumulation was assessed by immunostaining in small intestinal tissues from Ripk3 WT and Ripk3 iECKO mice following 24 hr I/R injury and clodronate-liposome treatment. ( I ) CD45 + cells, as in G, were quantified from multiple confocal Z-stack images taken from each mouse; CD45 + expression area was normalized to total cell nuclear area in small intestines. Individual data points are averages of 3–4 imaged and quantified sections per mouse (n=3/group). Data represent mean ± SEM. P values were determined by two-tailed Student’s t -test ( B , D , E and I ) and two-way ANOVA ( G ).

Article Snippet: The MS1 adult murine pancreatic EC line (ATCC; #CRL 2279) was cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco; #11960–044) supplemented with 10% fetal bovine serum (FBS) (Fisher Scientific; 3SH30910.03) and 1% antibiotics (streptomycin/amphotericin; Sigma; #A5955).

Techniques: Activation Assay, Permeability, Immunostaining, Expressing, Labeling, Incubation, Imaging, Fluorescence, Control, Two Tailed Test

Journal: Nature Communications

Article Title: Rapid amide ligation between α-halo acylsilanes and amines under aqueous conditions

doi: 10.1038/s41467-025-65617-y

Figure Lengend Snippet: LC-MS and SDS-PAGE gel analysis of proteins modified. Reaction of lysozyme ( A , B ), chymotrypsinogen A ( C ), myoglobin ( D ) and ribonuclease A ( E ) with α-halo acylsilanes in DMSO/NH 4 OAc (20 mM) = 4/1 at 25 °C for 1 h, independent replicates (n = 4) of SDS-PAGE gel analysis were performed in ( B ), with similar results. The mass spectrometry data had not been deconvoluted, and the number of added ions had been marked in the figure. (+1 mod. = singly modified protein, +2 mod. = doubly modified protein, etc.).

Article Snippet: Ribonuclease A, from bovine pancreas was purchased from MedChemExpress (MCE).

Techniques: Liquid Chromatography with Mass Spectroscopy, SDS Page, Modification, Mass Spectrometry