Journal: Cell proliferation

Article Title: Single-cell transcriptomic analysis of the senescent microenvironment in bone metastasis.

doi: 10.1111/cpr.13743

Figure Lengend Snippet: FIGURE 4 CD4Tstr cells exhibit pronounced senescent features in the bone-metastatic microenvironment. (A) UMAP plot of 45,603 subclustered T/NK cells coloured by annotated cell types. (B) Heatmap illustrating the expression of selected signatures in annotated subtypes. (C) Boxplot indicating the proportion of annotated cell types in PT and BM. (D-E) Representative immunofluorescence images (D) and boxplot (E) showing the expression level of CD4Tstr cells (positive for CD3D, CD4, and HSP70) in PT and BM. Scale bar: 20 μm (200) and 10 μm (400). (F) Kaplan–Meier plot showing the difference in survival probability (%) in patients with high versus low scores of the CD4Tstr signature according to the optimal cutoff based on the TCGA-NSCLC cohort. (G) Dot plot displaying the expression of selected genes across T-cell subclusters and sample groups. (H) Violin-boxplots displaying senescent scores in CD4Tstr cells from PT and BM. (I) Spearman correlation of the SenMayo signature with the angiogenesis signature by cells (left panel) and by samples (right panel) in CD4Tstr cells. (J) Spearman correlation of the Fridman_up signature with the angiogenesis signature by cells (left panel) and by samples (right panel) in CD4Tstr cells. PT, primary tumour; BM, bone metastases; NSCLC, non-small cell lung cancer.

Article Snippet: After deparaffinization, rehydration, and antigen retrieval by microwave, the tissue slides were incubated with primary antibodies targeting CD3D (1:200; 16669-1-AP; Proteintech, Wuhan, China), CD4 (1:200; 67786-1-Ig; Proteintech), CD8A (1:400; 66868-1-Ig; Proteintech), HSP70 (1:100; 66183-1-Ig; Proteintech), SOX18 (1:100; sc-166025; Santa Cruz Biotechnology, Dallas, TX, USA), CD31 (1:200; 66065-2-Ig; Proteintech), SERPINE1 (1:100; sc5297; Santa Cruz Biotechnology), KRT19 (1:200; 10712-1-AP; Proteintech) and SPP1 (1:100; 22952-1-AP; Proteintech).

Techniques: Expressing, Immunofluorescence

Journal: Nature Communications

Article Title: The phosphatase DUSP22 inhibits UBR2-mediated K63-ubiquitination and activation of Lck downstream of TCR signalling

doi: 10.1038/s41467-024-44843-w

Figure Lengend Snippet: a Identification of CUL1 as an UBR2-interacting protein by mass spectrometry-based proteomics. Identified CUL1 peptides were shown. b Immunoblotting of CUL1, Flag-tagged UBR2, Myc-tagged (DUSP22), and GAPDH proteins in Jurkat T cells co-transfected with Myc-CUL1, Flag-UBR2, and Myc-DUSP22 plasmids. c Immunoblotting of the endogenous CUL1, Flag-tagged UBR2, Myc-tagged DUSP22, and vinculin proteins in HEK293T cells co-transfected with Flag-UBR2, Myc-DUSP22, and CUL1 shRNA plasmids. d CUL1 knockdown inhibited DUSP22-induced UBR2 ubiquitination. Immunoprecipitation and immunoblotting analysis of Lys48-linked ubiquitination of UBR2, Flag-tagged UBR2, Myc-tagged DUSP22, and endogenous CUL1 proteins were performed using the lysates of HEK293T cells co-transfected with Flag-UBR2, Myc-DUSP22, and CUL1 shRNA #3 plasmids. The transfected cells were treated with 25 µM MG132 for 4 h. Arrow, UBR2 protein; asterisk, degraded UBR2 protein. e βTrCP overexpression plus suboptimal DUSP22 (0.8 μg) induced UBR2 degradation. Immunoblotting of Flag-tagged UBR2, Myc-tagged DUSP22, and tubulin proteins in Jurkat T cells co-transfected with Flag-UBR2 and Myc-DUSP22 plus different amounts (0.8 μg, 1.6 μg) of Flag-βTrCP plasmids. f UBR2 interacted with βTrCP. Immunoprecipitation and immunoblotting of Flag-tagged UBR2 with Myc-tagged-βTrCP proteins were performed using the lysates of HEK293T transfected with Flag-UBR2 and Myc-βTrCP plasmids. Anti-vinculin immunoblotting was performed by reprobing the anti-Flag (UBR2) immunoblot membrane. g Confocal microscopy analyses of PLA for the interaction between Flag-tagged UBR2 and Myc-tagged βTrCP proteins in HEK293T cells co-transfected with Flag-UBR2, Myc-βTrCP, and either GFP-DUSP22 or GFP-DUSP22 (C88S) plasmids. Red fluorescence represents the interactions of UBR2 with βTrCP proteins. Images were captured with 400X original magnification. Cell nuclei were stained with DAPI. Scale bar, 50 μm. h Immunoblotting of endogenous UBR2, βTrCP, or CUL1 proteins in Jurkat T cells transfected with scramble shRNAs, βTrCP shRNAs #1, or CUL1 shRNAs #3. T cells were stimulated with anti-CD3 antibody for indicated time periods. i CUL1-βTrCP E3 ligase complex induced UBR2 ubiquitination in vitro. Recombinant His-ubiquitin, E1 (UBA1), E2 (top panel, UBE2D3; bottom panel, CDC34), ATP, and SKP1-CUL1-βTrCP-RBX1 complex were co-incubated in the ubiquitination buffer with Flag-tagged UBR2 E3 ligase-inactive mutant (C1210/1213A) proteins.

Article Snippet: Anti-CD3ζ (Y142) antibody (Cat A02421Y142, Lot A02421Y142) was purchased from Boster Biotechnology.

Techniques: Mass Spectrometry, Western Blot, Transfection, shRNA, Knockdown, Ubiquitin Proteomics, Immunoprecipitation, Over Expression, Membrane, Confocal Microscopy, Fluorescence, Staining, In Vitro, Recombinant, Incubation, Mutagenesis

Journal: Nature Communications

Article Title: The phosphatase DUSP22 inhibits UBR2-mediated K63-ubiquitination and activation of Lck downstream of TCR signalling

doi: 10.1038/s41467-024-44843-w

Figure Lengend Snippet: a Schematic diagram of the murine UBR2 wild-type (WT) alleles and the targeted UBR2 mutant alleles. P1 and P2, the primers for PCR. b PCR analyses of wild-type and UBR2 mutant alleles using genomic DNAs from mouse tails. The PCR product of the upper band (579 bp) denotes the wild-type allele, and the lower band (511 bp) denotes the UBR2 mutant allele. c Immunoblotting analyses of UBR2 protein levels in T cells of wild-type or UBR2 knockout (KO) mice. d UMAP plot showed dimensional reduction of the distribution of 3061 cells. T cells derived from each group are shown in different colors (blue: wild-type; red: UBR2 knockout). e UMAP plot showed seven major clusters of the 3061 individual T cells. The number denotes the cluster frequency in parent population. f Volcano plot showed the differential expression transcripts between UBR2 knockout and wild-type T cells. Fold change represents the gene expression in UBR2 knockout T cells versus wild-type T cells. g Violin plots showed the expression levels of IFN-γ, TNF-α, IL-2, IL-4, and IL-6 genes in wild-type T cells under different UBR2 expression levels. The mRNA levels of UBR2 and cytokines were detected as unique molecular identifier (UMI) counts. h ELISAs of IFN-γ, TNF-α, and IL-17A levels in culture supernatants from peripheral blood T cells treated with plate-bound anti-CD3 plus anti-CD28 costimulation for 72 h. Total murine primary T cells were used in Fig. 5c-h.

Article Snippet: Anti-CD3ζ (Y142) antibody (Cat A02421Y142, Lot A02421Y142) was purchased from Boster Biotechnology.

Techniques: Mutagenesis, Western Blot, Knock-Out, Derivative Assay, Quantitative Proteomics, Gene Expression, Expressing

Journal: Nature Communications

Article Title: The phosphatase DUSP22 inhibits UBR2-mediated K63-ubiquitination and activation of Lck downstream of TCR signalling

doi: 10.1038/s41467-024-44843-w

Figure Lengend Snippet: a Immunoprecipitations of the endogenous Lck with either UBR2 or DUSP22 proteins in the lysates of anti-CD3-stimulated murine primary T cells. b TCR signaling stimulated Lys63-linked ubiquitination and Tyr394 phosphorylation of Lck in murine primary T cells. Endogenous Lck immunocomplexes in the lysates of anti-CD3-stimulated T cells were immunoprecipitated with anti-Lck antibody and then subjected to immunoblotting. c Confocal microscopy analyses of PLA for the ubiquitinated and phosphorylated Lck in TCR-stimulated T cells of wild-type (WT) or UBR2 knockout mice using anti-Lck antibody plus either anti-ubiquitin (Lys63) or anti-phospho-Lck (Tyr394) antibodies. Red fluorescence represents the endogenous Lck proteins containing Lys63-linked ubiquitination or Tyr394 phosphorylation. Cell nuclei were stained with DAPI. d UBR2 induced Lck ubiquitination in vitro. Recombinant His-ubiquitin, E1 (UBE1), E2 (UBE2D3), Flag-Lck, and ATP were co-incubated in the ubiquitination buffer with E3 (Flag-tagged UBR2 or Flag-tagged UBR2 ligase-inactive mutant (C1210/1213A)) proteins. e The Lys99 and Lys276 residues were identified as ubiquitination sites of Lck by mass spectrometry analysis. K(ub), ubiquitinated lysine residue. f Immunoprecipitations of ubiquitinated Flag-tagged Lck with Myc-tagged UBR2 proteins in the lysates of indicated HEK293T transfectants. Flag-tagged Lck proteins were immunoprecipitated with anti-Flag antibody and then immunoblotted with anti-ubiquitin (Lys63) antibody or anti-Flag antibody. g Immunoprecipitation and immunoblotting analysis of Flag-tagged Lck with Tyr394 phosphorylated Lck proteins in the lysates of Jurkat T cells transfected with Flag-Lck (WT) or Flag-Lck (K99/276R) plasmid. h Confocal microscopy analyses of PLA for the ubiquitinated Lck and Tyr394-phosphorylated Lck in TCR-stimulated Jurkat transfectants using anti-Lck plus anti-ubiquitin (Lys63) and anti-phospho-Lck (Tyr394) antibodies, respectively. Red fluorescence represents the endogenous Lck proteins containing Lys63-linked ubiquitination or Tyr394 phosphorylation. Cell nuclei were stained with DAPI. i Double mutations (K99/276R) of Lck inhibited phosphorylation of Lck at Tyr394 residue by in vitro kinase assay. j In vitro ubiquitin E3 ligase assay in combination with kinase assay showed that ubiquitination-deficient Lck inhibited Lys63-linked polyubiquitination-induced trans-autophosphorylation of Lck by UBR2. Arrow, intact UBR2 protein; asterisk, degraded UBR2 protein.

Article Snippet: Anti-CD3ζ (Y142) antibody (Cat A02421Y142, Lot A02421Y142) was purchased from Boster Biotechnology.

Techniques: Ubiquitin Proteomics, Phospho-proteomics, Immunoprecipitation, Western Blot, Confocal Microscopy, Knock-Out, Fluorescence, Staining, In Vitro, Recombinant, Incubation, Mutagenesis, Mass Spectrometry, Residue, Transfection, Plasmid Preparation, Kinase Assay

Journal: Nature Communications

Article Title: The phosphatase DUSP22 inhibits UBR2-mediated K63-ubiquitination and activation of Lck downstream of TCR signalling

doi: 10.1038/s41467-024-44843-w

Figure Lengend Snippet: a Immunoblotting of phospho-Tyr394 Lck, Lck, and GAPDH proteins from purified peripheral blood murine T cells of wild-type (WT), DUSP22 knockout (KO), or DUSP22/UBR2 double knockout (dKO) mice upon anti-CD3 stimulation. Quantification of p-Lck proteins (normalized to Lck proteins) is shown at the bottom of p-Lck panel. b , c Confocal microscopy analyses of PLA for ubiquitinated Lck ( b ) and Tyr394 phosphorylated Lck ( c ) proteins in TCR-stimulated T cells of wild-type, DUSP22 knockout, or DUSP22/UBR2 double knockout (dKO) mice using anti-Lck antibody plus anti-ubiquitin (Lys63) antibody ( b ) and anti-phospho-Lck (Tyr394) antibody ( c ), respectively. Red fluorescence represents the endogenous Lck proteins containing Lys63-linked ubiquitination or Tyr394 phosphorylation. Cell nuclei were stained with DAPI. The red signal intensity was plotted in the lower panels. d Serum levels of IFN-γ, TNF-α, and IL-17A in wild-type, DUSP22 knockout (KO), and DUSP22/UBR2 double knockout (dKO) mice were determined by ELISAs. e Hematoxylin and eosin (H&E)-stained sections of the kidney, liver, and lung from 10- to 11-month-old wild-type, DUSP22 knockout (KO) and DUSP22/UBR2 double knockout (dKO) mice. Scale bar, 50 μm (kidney and liver) and 100 μm (lung). f Western blotting analysis of UBR2, p-Lck, and DUSP22 proteins in peripheral blood T cells of human SLE patients and healthy controls (HC). Anti-GAPDH immunoblotting was performed by reprobing the anti-Lck immunoblot membrane. g , h Confocal microscopy analyses of PLA for the interaction between the endogenous UBR2 and Lck proteins or Lys63-linked ubiquitination of Lck in peripheral blood T cells of a representative human SLE patient and a representative healthy control (HC). T-cell nuclei were stained with DAPI. i ELISAs of IFN-γ, TNF-α, and IL-17A levels in culture supernatants from MOG-restimulated T cells. Mice were immunized with MOG peptide emulsified in CFA, followed by injection of pertussis at day 0, 1, and 2. To determine the activation of Ag-specific T cells, T cells from lymph nodes of the immunized mice were cultured in the presence of 0, 20, and 50 μg/ml MOG for 72 h, and cytokine levels of IFN-γ, TNF-α, and IL-17A were analyzed by ELISA. n.s., not significant.

Article Snippet: Anti-CD3ζ (Y142) antibody (Cat A02421Y142, Lot A02421Y142) was purchased from Boster Biotechnology.

Techniques: Western Blot, Purification, Knock-Out, Double Knockout, Confocal Microscopy, Ubiquitin Proteomics, Fluorescence, Phospho-proteomics, Staining, Membrane, Control, Injection, Activation Assay, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: Inflammatory cytokines in small intestinal mucosa of patients with potential coeliac disease

doi: 10.1046/j.1365-2249.2002.01798.x

Figure Lengend Snippet: Monoclonal mouse anti-human antibodies (MoAbs) used in this study

Article Snippet: CD3 , CD3-T cell receptor , 1:200 , Becton-Dickinson, San Jose, CA, USA.

Techniques: