mmol  (Chem Impex International)

Journal: bioRxiv

Article Title: B lymphocytes acquire myeloid and autoimmune phenotypes via the downregulation of lymphocyte-specific protein-1

doi: 10.1101/2024.06.28.600734

Figure Lengend Snippet: a, Heatmap displaying the DEGs between WT and Lsp1 -/- B cells stimulated with αIgM or LPS for 4 hours. Red and blue indicate up- and downregulation, respectively (left panel). The right panel shows the hematopoietic cell markers upregulated by Lsp1 deficiency. b , Upregulation of myeloid gene expression in Lsp1 -/- B cells, as determined by qRT‒PCR; the fold changes were calculated using the 2 -ΔΔCt method. c, Percentage of MPO + cells in splenic B cells freshly isolated from WT and Lsp1 -/- mice (n=20 per group), as determined by immunofluorescence staining with anti-B220 (green) and anti-MPO (red) Abs. d - i, Splenic B cells isolated from WT and Lsp1 -/- mice were stimulated with αIgM or LPS for 24 hours ( d-f ), 7 days ( g ), or 5 days ( h,i ). d , Immunoblot (left panel) and ELISA (right panel, n=10 per group) analyses of MPO expression. e , Intracellular ROS production (n=7 per group) by flow cytometry using H 2 DCFDA. f , Production of IL-1β and TNF-α (n=9∼10) by ELISA. g , Percentage of CD11c + cells in WT and Lsp1 -/- B cells (n=9∼10) by flow cytometry. A representative zebra plot for CD11c + cells (red dotted box) is presented on the left. h , Immunoblotting for Blimp1 and Xbp1, which are representative TFs involved in B-cell differentiation. i , ELISA for IgG, IgG2c, and IgG3 produced by B cells stimulated with LPS (n=3 per group). j,k, LSP1 -regulated gene expression and IgG production in human B cells. Sorted human B cells were electroporated with LSP1 siRNA ( j ) or an LSP1-EGFP plasmid ( k ). After 24 hours, the mRNA expression levels of LSP1 , MPO , IL1B , and CYBB in the cells were determined via qRT‒PCR. IgG production by B cells stimulated with αIgM, CD40L, and CpG for 5 days was determined via ELISA. l, GSEA plots demonstrating significant enrichment between the Lsp1 -regulated genes in Clusters 1 and 2 and the genes (up- and downregulated) defining CD11b + B cells, CD11c + B cells, or ABCs. NES=normalized enrichment score. The data in the bar graphs are presented as the means ± SDs of at least two independent experiments. P values were determined by multiple unpaired two-tailed t tests ( b to g ), two-way ANOVA with Sidak’s multiple comparisons ( i , right panels in j and k ), and the Mann‒Whitney U or unpaired t test (left panels in j and k ). * P < 0.05; ** P < 0.01; *** P < 0.001; and **** P < 0.0001.

Article Snippet: For gene knockdown, human LSP1 siRNA (Santa Cruz Biotechnology, #sc-42899), ETS1 siRNA (Santa Cruz Biotechnology #sc-29309), or mouse PKCβ siRNA (Santa Cruz Biotechnology, #sc-36255) was electroporated into B cells using an Amaxa Nucleofector (Lonza) and incubated as indicated.

Techniques: Gene Expression, Isolation, Immunofluorescence, Staining, Western Blot, Enzyme-linked Immunosorbent Assay, Expressing, Flow Cytometry, Cell Differentiation, Produced, Plasmid Preparation, Two Tailed Test

Journal: bioRxiv

Article Title: B lymphocytes acquire myeloid and autoimmune phenotypes via the downregulation of lymphocyte-specific protein-1

doi: 10.1101/2024.06.28.600734

Figure Lengend Snippet: a, Venn diagram displaying transcription factors (TFs) that have a high potential to regulate the 369 upregulated DEGs in Lsp1 -/- B cells with or without αIgM or LPS treatment (left panel). The bar graph on the right depicts the numbers of C/EBP target genes among the DEGs in Lsp1 -/- B cells. b, PPI network representing the interactions between C/EBPα/β and the 31 C/EBP target genes involved hematopoietic cell lineage markers, cytokines/chemokines, and phagocytosis. The color of the nodes indicates the fold change in the genes affected by Lsp1 deficiency. c, d, Quantitative RT‒PCR analysis of Cebpa and Cebpb mRNA expression (n=4∼7; c ) and immunoblotting for C/EBPβ protein expression ( d ). Lsp1 -/- versus WT B cells were stimulated with αIgM or LPS for 4 hours ( c ) or for 24 hours ( d ). e, f, Lsp1 -/- B cells were transduced with control or Cebpb shRNAs, and the cells were then stimulated with αIgM or LPS for 24 hours for C/EBPβ immunoblotting ( e ) and MPO and TNF-α ELISAs (n=4; f ). g, Control or Cebpb shRNA-treated Lsp1 -/- B cells were stimulated with αIgM or LPS for 5 days, after which Blimp1, IRF4, and Xbp1 expression was determined via immunoblot assay. h, Immunoblots of phosphorylated PKCβ (pPKCβ) in Lsp1 -/- versus WT B cells. Splenic B cells were stimulated with αIgM or LPS for 5 minutes. i, j, Effects of PKCβ inhibition on C/EBPβ and IL-1β expression in Lsp1 -/- B cells stimulated with αIgM or LPS. ( i ) Lsp1 -/- B cells were pretreated with the PKCβ inhibitor ruboxistaurin (4 μM) for 1 hour and stimulated with αIgM or LPS for 24 hours. ( j) Lsp1 -/- B cells were stimulated with αIgM or LPS for 24 hours and then electroporated with control or Prkcb siRNA for an additional 24 hours. The expression levels of C/EBPβ, its target gene IL-1β, and PKCβ were determined via an immunoblot assay. k, Proposed model for the dual regulation of B-cell functions by LSP1. The data in the bar graphs are presented as the means ± SDs of at least three independent experiments. P values were determined by multiple unpaired t tests ( c ) and paired t tests ( f ). * P < 0.05; ** P < 0.01.

Article Snippet: For gene knockdown, human LSP1 siRNA (Santa Cruz Biotechnology, #sc-42899), ETS1 siRNA (Santa Cruz Biotechnology #sc-29309), or mouse PKCβ siRNA (Santa Cruz Biotechnology, #sc-36255) was electroporated into B cells using an Amaxa Nucleofector (Lonza) and incubated as indicated.

Techniques: Expressing, Western Blot, Transduction, Control, shRNA, Inhibition

Journal: bioRxiv

Article Title: B lymphocytes acquire myeloid and autoimmune phenotypes via the downregulation of lymphocyte-specific protein-1

doi: 10.1101/2024.06.28.600734

Figure Lengend Snippet: a, Flow cytometry was used to determine LSP1 expression levels in the peripheral B cells of healthy donors (HD, n=20) and SLE patients (n=18): MFI=mean fluorescence intensity. Disease activity was evaluated by the SLEDAI. b, LSP1 expression in cultured B cells from HDs (n=4) versus SLE B cells (n=4∼5) determined by flow cytometry. B cells were stimulated with αIgM, CD40L, and CpG for 3 days. c, qRT‒PCR analysis of ETS1 mRNA expression in B cells from HDs (n=18) and SLE patients (n=17, left panel) and its correlation with LSP1 mRNA expression in HDs (black circle) and SLE patients (blue circle, right panel). d,e, Regulation of LSP1 expression by ETS1. d , ETS1 and LSP1 mRNA expression in human B cells transfected with ETS1 siRNA, as evaluated by qRT‒PCR (n=4). e , qRT‒PCR analysis of Ets1 and Lsp1 mRNA in B cells from WT ( Ets1 fl/fl , n=8) and Ets1 -/- ( Ets1 fl/fl C d19 Cre/- , n=4) mice. f, LSP1 expression in WT B cells (n=6∼7) stimulated with αIgM and LPS for 5 days in the presence or absence of (pretreated) 20 μM hydralazine, 20 μM procainamide, or 1 μM 5-azacytidine, as examined by flow cytometry. g, TEM images of B cells from HDs and SLE patients. h, i, Immunofluorescence staining of MPO in HDs and SLE B cells using anti-MPO (red) and anti-CD19 (green) Abs and DAPI (blue). h , Representative images of MPO + B cells are indicated by white arrows. i , Percentage of MPO + B cells determined by analyzing 6 fields per sample (n=5 for HDs, n=4 for SLE patients). j, qRT‒PCR and ELISA results for MPO. Left: MPO mRNA expression in HDs (n=17) versus SLE B cells (n=15). Right: ELISA for MPO production by B cells from HDs (n=7) and SLE patients (n=5) activated with the indicated stimuli for 3 days. k, Flow cytometry analysis of LSP1 and MPO expression in CD19 + B cells from HDs (black circle, n=4) and SLE patients (blue circle, n=7). l, Left: LSP1 signature in SLE patients and its correlation with the type I IFN signature: n=24 for the low IFN signature, n=75 for the high IFN signature, and n=181 for HD. Bulk RNA-seq data from SLE PBMCs were obtained from a public database (GSE72509). Right: Estimated population size of monocytic cells by cellular deconvolution analysis. Each dot represents an SLE patient or HD. m , LSP1 signature score in B cells from SLE patients versus HDs determined by pseudobulk RNA-seq analysis (GSE174188; s ee Methods ). n, Uniform Manifold Approximation and Projection (UMAP) plot visualizing the B-cell subsets with high LSP1 scores (>0.8) in SLE patients. The data in the bar graphs are presented as the means ± SDs of at least two independent experiments. P values were determined by the Mann‒Whitney U test (left panels in a , c , and j ; all data in i ); one-way ANOVA with Dunnett’s multiple comparisons (right panel in a ); two-way ANOVA with Sidak’s, Dunnett’s multiple comparison or multiple t test ( b , d - f ; right panel in j ); and the Spearman correlation test (right panels in c and k ). * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: For gene knockdown, human LSP1 siRNA (Santa Cruz Biotechnology, #sc-42899), ETS1 siRNA (Santa Cruz Biotechnology #sc-29309), or mouse PKCβ siRNA (Santa Cruz Biotechnology, #sc-36255) was electroporated into B cells using an Amaxa Nucleofector (Lonza) and incubated as indicated.

Techniques: Flow Cytometry, Expressing, Fluorescence, Activity Assay, Cell Culture, Transfection, Immunofluorescence, Staining, Enzyme-linked Immunosorbent Assay, RNA Sequencing, Comparison

Journal: MycoKeys

Article Title: Two new species and a new record of Hypoxylaceae ( Xylariales , Ascomycota ) from Mexico

doi: 10.3897/mycokeys.111.133046

Figure Lengend Snippet: Hypoxylon xmatkuilense sp. nov a general view of stromata b perithecia obovoid c KOH-extractable pigments d straight germ slit on the convex side e perispore dehiscent 10% KOH.

Article Snippet: Hypoxylon hinnuleum , ATCC 36255 , USA (T) , MK287537 , MK287549 , MK287562 , MK287575 , .

Techniques:

Journal: MycoKeys

Article Title: Two new species and a new record of Hypoxylaceae ( Xylariales , Ascomycota ) from Mexico

doi: 10.3897/mycokeys.111.133046

Figure Lengend Snippet: Hypoxylon bellicolor a general view of stromata b pruinose surface c KOH-extractable pigments d perithecia ovoid e asci f perispore dehiscent 10% KOH with coil-like ornamentation.

Article Snippet: Hypoxylon hinnuleum , ATCC 36255 , USA (T) , MK287537 , MK287549 , MK287562 , MK287575 , .

Techniques: