Journal: iScience

Article Title: PTEN acts as a crucial inflammatory checkpoint controlling TLR9/IL-6 axis in B cells

doi: 10.1016/j.isci.2024.110388

Figure Lengend Snippet: Ablation of PTEN in mature B cells led to early death in mice (A) FACS profiles of B220 versus CD5 in gated lymphocytes (left), and CD21 versus CD23 in CD5 + -gated B cells (right), from the spleens of CD23-control and CD23-cKO mice at 8–12 weeks of age. (B) Histograms showing the percentages of IM, FO, MZ, and B1a B cells in spleens from mice in (A) ( n = 3 male plus 2 female mice/group). (C) Representative images of spleens from the mice in (A). (D) Cell counts of total splenocytes (left) and splenic B cells (right) from the mice in (A). (E) Kaplan-Meier curves of female CD23-cKO ( n = 20) and female CD23-control ( n = 15) mice. (F) Representative images of spleens, mesenteric lymph nodes (MLNs), and axillary lymph nodes (ALNs) resected from the indicated mice at 34 weeks. (G) Cell counts of splenocytes (left) and splenic B cells (right) in the indicated mice ( n = 3–6 male mice/group) at 8 weeks or >16 weeks. (H) Levels of total IgM, total IgG1, and anti-nuclear and anti-dsDNA antibodies in the serum of the indicated mice ( n = 3–12 male mice/group) at 8–12 weeks or >16 weeks determined by ELISA. The samples were compared using unpaired two-tailed t test; ∗, p < 0.05; ∗∗, p < 0.005; ∗∗∗, p < 0.0005, and data are presented as mean ± SEM.

Article Snippet: Mouse anti-dsDNA ELISA Kit , Fujifilm-Wako-Shibayagi , Cat#AKRDD-061.

Techniques: Control, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: iScience

Article Title: PTEN acts as a crucial inflammatory checkpoint controlling TLR9/IL-6 axis in B cells

doi: 10.1016/j.isci.2024.110388

Figure Lengend Snippet: Loss of PTEN in B2 B cells promotes TLR9-mediated IL-6 production (A) Overlaid curves showing the expression levels of IL-6, IL-9, IL-17, CCL5, G-CSF, IL-4, IL-10, IFN-γ, and TNF-α in splenic B cells from the indicated mice ( n = 3 male mice/group) at 25–33 weeks was determined by flow cytometry. Cells were left unstimulated or stimulated with CpG, LPS, P+I, CpG+P+I, or LPS+P+I, in vitro for 24 h. (B) FACS profiles of IL-6 versus CD19 in splenic B cells from the indicated mice ( n = 9 male mice/group) at 10 w. (C) Percentages of IL-6 + B cells in splenic B cells as described in (B). (D) Histograms showing IL-6 produced by FO (left) and MZ (right) B cells, which were left unstimulated (resting) or stimulated with CpG, CL307, LPS, and poly(I:C) in vitro for 24 h, as determined by ELISA. Each group containing 3 male mice. (E) Histograms showing IL-6 produced by FO (left), MZ (middle), and B1a (right) B cells from the indicated mice that were left untreated (resting) or stimulated with CpG in vitro for 24 h, as determined by ELISA. Each group containing 4 male mice. (F) Histograms showing IL-6 produced by FO (left) and MZ (right) B cells, which were left untreated (resting) or stimulated with CpG in the absence or presence of inhibitors (inh.) targeting NF-κB p65 or p50, respectively, for 24 h, as determined by ELISA. Each group containing 4 male mice. (G) Kaplan-Meier curves of female CD23-control mice (black line, n = 16), female CD23-cKO mice (red line, n = 24), and female CD23-cKO/IL-6 −/− double knockout mice (blue line, n = 19). The samples were compared using an unpaired two-tailed t test; ∗, p < 0.05; ∗∗, p < 0.005; ∗∗∗, p < 0.0005, and the data are presented as mean ± SEM.

Article Snippet: Mouse anti-dsDNA ELISA Kit , Fujifilm-Wako-Shibayagi , Cat#AKRDD-061.

Techniques: Expressing, Flow Cytometry, In Vitro, Produced, Enzyme-linked Immunosorbent Assay, Control, Double Knockout, Two Tailed Test

Journal: iScience

Article Title: PTEN acts as a crucial inflammatory checkpoint controlling TLR9/IL-6 axis in B cells

doi: 10.1016/j.isci.2024.110388

Figure Lengend Snippet: PTEN deletion in B2 B cells resulted in PI(3)P accumulation and enhanced TLR9 endosomal localization (A) Confocal images showing the levels of PI(3)P, LAMP-1, and DAPI in the indicated B cells. Scale bars, 5 μm. (B) Histograms showing the intensity of PI(3)P and LAMP-1 in the indicated B cells calculated using the mean fluorescence intensity (MFI) from 36 to 186 individual cells per group as described in (A). (C) Confocal images showing the levels of PI(3)P, LAMP-1, and DAPI in the indicated B cells. Scale bars, 5 μm. (D) Histograms showing the intensity of PI(3)P and LAMP-1 in the indicated B cells calculated using MFI from 56 individual cells per group as described in (C). (E) Confocal images showing the interaction between TLR9 and PI(3)P by PLA in the indicated B cells. Scale bars, 5 μm. (F) Histogram showing the quantified PLA signals of TLR9/PI(3)P in the indicated B cells calculated from 35 to 42 individual cells per group as described in (E). (G) A diagram illustrating phosphatidylinositol signaling regulated by PI3K and lipid phosphatases. The accumulation of PI(3)P in PTEN-deficient B cells is indicated by a red arrow. (H) Histograms showing IL-6 produced by FO (left) and MZ (middle) B cells from the indicated mice that were left unstimulated or stimulated with CpG in the absence or presence of inhibitors (inh.) targeting PIK3C3 or INPP4 in vitro for 24 h, as determined by ELISA. Each group containing 4 male mice. The samples were compared using an unpaired two-tailed t test; ∗, p < 0.05; ∗∗, p < 0.005; ∗∗∗, p < 0.0005, and the data are presented as mean ± SEM.

Article Snippet: Mouse anti-dsDNA ELISA Kit , Fujifilm-Wako-Shibayagi , Cat#AKRDD-061.

Techniques: Fluorescence, Produced, In Vitro, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: iScience

Article Title: PTEN acts as a crucial inflammatory checkpoint controlling TLR9/IL-6 axis in B cells

doi: 10.1016/j.isci.2024.110388

Figure Lengend Snippet: scRNA-seq analysis revealed that endocytosis was essential for TLR9-mediated IL-6 (A) A diagram illustrating splenic B cells collected from the four mice strain and the treatments for scRNA-seq analysis. (B) Hallmark gene sets analysis showing 21 pathways upregulated and 16 pathways downregulated in the CpG-stimulated splenic B cells from CD23-cKO mice. (C) KEGG database analysis showing 28 pathways upregulated and 10 pathways downregulated in the CpG-stimulated splenic B cells from CD23-cKO mice. (D) Dotplot analysis showing selected gene clusters from (B) and (C) in B cells in the resting state, upon CpG-stimulation or upon CpG-stimulation in the presence of p38 inhibitor (p38i) from the indicated mice. (E) Histograms showing IL-6 produced by FO (left) and MZ (right) B cells, which were left unstimulated (−) or stimulated (+) with CpG for 24 h in the absence or presence of inhibitors (inh.) targeting caveolin 1 or clathrin, respectively, as determined by ELISA. Each group containing 2 male and 2 female mice. The samples were compared using an unpaired two-tailed t test; ∗, p < 0.05; ∗∗, p < 0.005; ∗∗∗, p < 0.0005, and the data are presented as mean ± SEM.

Article Snippet: Mouse anti-dsDNA ELISA Kit , Fujifilm-Wako-Shibayagi , Cat#AKRDD-061.

Techniques: Produced, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: iScience

Article Title: PTEN acts as a crucial inflammatory checkpoint controlling TLR9/IL-6 axis in B cells

doi: 10.1016/j.isci.2024.110388

Figure Lengend Snippet: Ectopic BCR expression in mb1-cKO B cells reconstitutes the expression of BANK1 and NF-κB p50 and elevates TLR9-mediated IL-6 production (A) FACS profiles showing the percentages of B220 + splenic B cells from the indicated mice. (B) Overlaid curves showing levels of surface IgM and IgD in IM, FO, and MZ B cells from the indicated mice. (C) Western blot analyses to show the expression of BANK1, NF-κB p105 and p50, p-p38, and β-actin, in splenic B cells from the indicated mice, which were left unstimulated (0) or stimulated with CpG for 5 and 15 min. Each group containing 3 male mice. (D) Overlaid curves showing p-p65 S536 signals by intracellular staining in splenic B cells from the indicated mice, which were left unstimulated (0) or stimulated with CpG for 1, 5, 15, and 30 min. Each group containing 3 male mice. (E) Histograms showing IL-6 produced by FO (left) and MZ (right) B cells from the indicated mice, which were left unstimulated (−) or stimulated with CpG for 24 h, as determined by ELISA. Each group containing 3 male mice. The samples were compared using an unpaired two-tailed t test; ∗, p < 0.05; ∗∗, p < 0.005; ∗∗∗, p < 0.0005, and the data are presented mean ± SEM.

Article Snippet: Mouse anti-dsDNA ELISA Kit , Fujifilm-Wako-Shibayagi , Cat#AKRDD-061.

Techniques: Expressing, Western Blot, Staining, Produced, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: iScience

Article Title: PTEN acts as a crucial inflammatory checkpoint controlling TLR9/IL-6 axis in B cells

doi: 10.1016/j.isci.2024.110388

Figure Lengend Snippet:

Article Snippet: Mouse anti-dsDNA ELISA Kit , Fujifilm-Wako-Shibayagi , Cat#AKRDD-061.

Techniques: Recombinant, Western Blot, Staining, In Situ, Antibody Labeling, Enzyme-linked Immunosorbent Assay, Software