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

Journal: Advanced Science

Article Title: PLAUR + Neutrophils Drive Anti‐PD‐1 Therapy Resistance in Patients with Hepatocellular Carcinoma by Shaping an Immunosuppressive Microenvironment

doi: 10.1002/advs.202507167

Figure Lengend Snippet: PLAUR inhibitor reverses the immunosuppressive phenotype of neutrophils and attenuates tumor progression. A) Flowchart depicting the screening strategy for small‐molecule compounds targeting PLAUR. B) The affinity of 40 candidate small‐molecule compounds targeting PLAUR detected by SPR analysis. C) Schematic diagram of the predicted docking structure of DAPTA and PLAUR. D) Cell viability of DAPTA detected in human neutrophils. E) Western blot analysis of PLAUR, phosphorylated and non‐phosphorylated NF‐κB expression in human neutrophils treated with DMSO or DAPTA (5 µ m ). F) quantitative PCR analysis of the indicated genes in DMSO and DAPTA treatment groups ( n = 3 per group). G) Flow cytometry analysis of CD206 and CD95 expression in DMSO and DAPTA treatment groups (n = 3 per group). H) Schematic showing the treatment plan and establishment of spontaneous HCC models in mice. I) Representative images of the spontaneous tumors at the study endpoint (5 mice per group). Scale bar: 1 cm. J) The maximum tumor volume of each group at the study endpoint ( n = 5 per group). K) Kaplan‐Meier survival curves for mice ( n = 5 per group). L) Immunofluorescence staining and statistical analysis of PLAUR, F4/80 and CD8 in the indicated groups ( n = 5 per group). Scale bars: 100 µm. M,N) Flow cytometry analysis of Ly6G + CD206 + neutrophils, CD11b + F4/80 + macrophages and CD3 + CD8 + T cells in the indicated groups ( n = 5 per group). The data are presented as the means ± SDs. * P < 0.05, ** P < 0.01, and *** P < 0.001, Student's t test.

Article Snippet: For PLAUR inhibitor treatment, mice were treated intraperitoneally with D‐Ala‐peptide T‐amide (DAPTA) (MedChemExpress, 100 μg per mouse) following the schedule shown in Figures and .

Techniques: Western Blot, Expressing, Real-time Polymerase Chain Reaction, Flow Cytometry, Immunofluorescence, Staining