Journal: JCI Insight

Article Title: Platelets impair the resolution of inflammation in atherosclerotic plaques in insulin-resistant mice after lipid lowering

doi: 10.1172/jci.insight.193593

Figure Lengend Snippet: ( A ) Study design. Eight-week-old Ldlr –/– male mice fed a high-fat, high-cholesterol (HFHC) diet for the whole duration of the study (23 weeks). After the atherosclerosis progression period (obese baseline, Ob), mice were injected with ApoB ASO to lower lipids (lipid-lowering period). All lipid-lowered mice were split into 2 groups, injected either with isotype control antibody (IgG) or platelet depletion antibody (αCD42b) every 3 days for a total of 3 weeks. ( B ) Platelet counts in circulating blood of Ob, IgG, and αCD42b 3 days before harvest. ( C ) Blood glucose measurements (lean n = 16, Ob n = 47) and ( D ) area of curve (AOC) quantification of glucose tolerance tests (GTTs) from lean, Ob, IgG, and αCD42b mice after 16 weeks of HFHC diet during atherosclerosis progression. ( E ) Blood glucose measurements (Ob n = 3, IgG n = 4, αCD42b n = 4) and ( F ) AOC quantification of GTTs from Ob, IgG, and αCD42b mice after 22 weeks of HFHC diet during atherosclerosis resolution. Mean fluorescent intensity (MFI) of platelet ( G ) JonA and ( H ) P-selectin in nonstimulated (NS) samples and upon 100 μM PAR4-AP agonist stimulation of lean and Ob circulating blood samples. ( I ) Mean platelet volume (MPV) from circulating blood of lean, Ob, and IgG-treated mice, 3 days before harvest. In C and E , error bars represent SD. Data were analyzed by Kruskal-Wallis with Dunn’s post hoc test ( B and F ), unpaired Mann-Whitney test ( C , D , and H ), unpaired Student’s t test ( G ), or ordinary 1-way ANOVA with Tukey’s multiple-comparison test ( I ). P values are shown in graphs.

Article Snippet: The whole blood was stained with anti–mouse JonA-PE (M023-2, Emfret Analytics) and anti–mouse P-selectin Alexa Fluor 647 (563674, BD Biosciences) and then treated with PAR4-AP platelet agonist (100 μM; MedChemExpress) for 15 minutes at room temperature.

Techniques: Injection, Control, MANN-WHITNEY, Comparison

Journal: The Journal of Cell Biology

Article Title: Assembly and phosphoregulatory mechanisms of the budding yeast outer kinetochore KMN complex

doi: 10.1083/jcb.202506015

Figure Lengend Snippet: Regulation of Mis12c Mtw1c auto-inhibition by Aurora B kinase and its effect on the interaction with CENP-C Mif2 . (A) Structural alignment of the crystal structure of K. lactis CENP-C Mif2 residues 4–36 ( Kl CENP-C Mif2_4-36 ) as bound to the Kl Mis12c Mtw1c head 1 domain (PDB: 5T59 ) to the experimental model of Sc Mis12c Mtw1c (this study). The models are colored by chain; Mis12 Mtw1 and Nnf1 are colored identically in 5T59 and our experimentally determined model. Kl CENP-C Mif2_4-36 is shown in space filling representation to highlight lack of steric clash, and overlapping binding site, with Dsn1 AI . Only Kl CENP-C Mif2_4-36 is shown. (B) SEC elution chromatograms of attempts to reconstitute the interaction between Knl1c:Mis12c Mtw1c complexes containing different Dsn1 N-terminal truncation and/or phosphomimetic mutants and CENP-C Mif2 residues 1–63 (CENP-C Mif2_1-63 ) indicated within red boxes. (C) Coomassie brilliant blue–stained SDS-PAGE gels of the chromatograms shown in B. (B and C) K HB-RWD M = full-length Dsn1. K HB-RWD M 2E = full-length Dsn1 containing Dsn1 S240E and Dsn1 S250E phosphomimetic mutations. K HB-RWD M Dsn1Δ257 = Dsn1 residues 1–257 are deleted (Dsn1 Δ1-257 ). K HB-RWD M Dsn1Δ226 = Dsn1 residues 1–226 are deleted (Dsn1 Δ1-226 ). K HB-RWD M Dsn1Δ226-2E = Dsn1 residues 1–226 are deleted and Dsn1 S240E and Dsn1 S250E phosphomimetic mutations (Dsn1 Δ1-226,S240E,S250E ). CENP-C Mif2_1-63 is indicated within a red box. (D) ITC data for (i) CENP-C Mif2_1-38 interactions with KM, and (ii) CENP-C Mif2_1-38 interactions with KM 2E . K D and n values are an average from three experiments (technical repeats). Source data are available for this figure: .

Article Snippet: CENP-U Ame1_1-33 (MDRDTKLAFRLRGSHSRRTDDIDDDVIVFKTPNW-amide), CENP-U Ame1_1-33-Mut (MDRDTKLAFRLRGSHSRRTDDIDDAAAVAKAPNW-amide), and CENP-C Mif2_1–38 (MDYMKLGLKSRKTGIDVKQDIPKDEYSMENIDDFFKDD-amide) peptides were synthesized by Alta BioScience.

Techniques: Inhibition, Binding Assay, Staining, SDS Page