Journal: Advanced Science

Article Title: PGRN Inhibits Early B‐cell Activation and IgE Production Through the IFITM3‐STAT1 Signaling Pathway in Asthma

doi: 10.1002/advs.202403939

Figure Lengend Snippet: PGRN Inhibition Enhances Early B‐Cell Activation by Modulating BCR Signaling A,B) TIRF microscopy (TIRFm) was employed to visualize alterations in PGRN and BCR levels in WT murine B cells after in vitro stimulation with membrane antigen. The Pearson correlation coefficients between BCR and PGRN expression levels are presented (B). The scale bar indicates 2.5 µm. C–J) Colocalization of BCR with pCD19, pY, pBTK, and pSHIP in splenic B cells from WT and PGRN KO mice after in vitro stimulation with soluble antigens was analyzed. Colocalization coefficients were calculated using NIS‐Elements AR software. The scale bar represents 2.5 µm. K–O) TIRFm analysis was conducted to assess the levels of BCR, pY, and pCD19 during spontaneous fluctuations in splenic B cells from WT and PGRN KO mice following in vitro stimulation with membrane antigen. Changes in the contact area and mean fluorescence intensities (MFIs) of BCR, pY, and pCD19 were quantified using TIRFm. The scale bar indicates 2.5 µm. P) Western blot analysis was performed to measure the levels of pBTK, BTK, pCD19, CD19, pSHIP‐1, SHIP‐1, and pY in B cells from WT and PGRN KO mice after in vitro stimulation with soluble antigen, using β‐ACTIN or total protein as loading controls. Representative blots from three independent experiments are shown. Q) Representative calcium flux flow cytometry plots from three independent experiments demonstrate the effects of in vitro treatment with F(ab') 2 Ig (M + G) on purified splenic B cells.

Article Snippet: Following incubation, the cells were fixed, permeabilized, and stained with a range of markers, including pCD19 (ab203615; Abcam), pBTK (5082S; Cell Signaling Technology), pY (05‐321; Merck‐Millipore), pSHIP (3941S; Cell Signaling Technology), and pWASP (A300‐205A; Bethyl Laboratories).

Techniques: Inhibition, Activation Assay, Microscopy, In Vitro, Membrane, Expressing, Software, Fluorescence, Western Blot, Flow Cytometry, Purification

Journal: Advanced Science

Article Title: PGRN Inhibits Early B‐cell Activation and IgE Production Through the IFITM3‐STAT1 Signaling Pathway in Asthma

doi: 10.1002/advs.202403939

Figure Lengend Snippet: PGRN Inhibits B‐cell Activation and IgE Production through the PGRN‐IFITM3‐STAT1 Signaling Pathway. A) The volcano plot illustrates the differential gene expression between WT and PGRN KO mice, displaying log2 fold change (log2FC) on the x ‐axis and –log10(Q) values on the y ‐axis for each gene, with colors representing different levels of significance. B) Differentially expressed (DE) genes overlapping with BCR‐associated genes from the KEGG database are highlighted. C) The mRNA levels of Ifitm3 in B cells from WT and PGRN KO mice are shown ( n = 9). D) Western blot analysis shows the expression of 14‐3‐3σ and IFITM3 in B cells from WT and PGRN KO mice, using β‐actin as a loading control. The blots shown are representative of three independent experiments. E‐F) Chromatin immunoprecipitation (ChIP) was performed on purified splenic B cells from WT mice, followed by DNA purification and PCR (left) and real‐time PCR (right) using primers specific to the 14‐3‐3σ promoter. G) A luciferase reporter assay was conducted in HEK293T cells to assess the regulation of 14‐3‐3σ promoter activity, utilizing the pGL3‐14‐3‐3σ promoter and pAd5‐E1‐CMV‐STAT1 plasmid. H) Western blot analysis of pSTAT5, STAT5, pIKKβ, IKKβ, pP65, and P65 in B cells from WT and PGRN KO mice following in vitro stimulation with soluble antigen, with representative blots from three independent experiments. I) Western blot analysis of pSTAT1 and pPI3K levels in B cells from WT and PGRN KO mice treated with caraphenol A, using total protein as a control; representative blots are from three independent experiments. J) Western blot analysis showing the expression of 14‐3‐3σ and IFITM3 in B cells from WT and PGRN KO mice treated with caraphenol A, with β‐actin or total protein as controls; blots represent three independent experiments. K,L) Flow cytometry data showing levels of IgE and IgG 1 in B cells from WT, PGRN KO, and PGRN KO mice treated with the STAT1 inhibitor Fludarabine after a 5‐day in vitro stimulation with anti‐CD40 antibody and IL‐4 ( n = 3). M,N) Flow cytometry data displaying IgE and IgG 1 levels in B cells from WT, PGRN KO, and PGRN KO mice treated with the IFITM3 inhibitor caraphenol A following a 5‐day in vitro stimulation with anti‐CD40 antibody and IL‐4 ( n = 5). O) Flow cytometric analysis of B‐cell subsets within PBMCs from healthy controls (HCs) and asthma patients, quantifying the percentages of naive B, memory B, transitional B, and plasmablast cells ( n = 6). P) A schematic overview of the house dust mite (HDM)‐induced asthma mouse model. Q) Proportions of B cells gated from O. R) Serum IgE levels in HCs and asthma patients ( n = 10). S) PBMCs from HCs and asthma patients were labeled with APC‐anti‐CD19 antibody, followed by biotin‐conjugated F(ab’) 2 Ig(M+G) and streptavidin, then stimulated at 37 °C. Following stabilization and permeabilization, mean fluorescence intensities (MFIs) of pY and pBTK were measured via flow cytometry ( n = 10). T) Western blot analysis shows PGRN, 14‐3‐3σ, and IFITM3 expression in WT and asthma model mice, as well as in HCs and asthma patients, with β‐actin as a control; blots are representative of three independent experiments. U) Western blot analysis of B cells from WT, asthma model mice, and asthma model mice treated with PGRN in vitro, assessing pPI3K, pCD19, pBTK, and 14‐3‐3σ expression, using β‐actin as the control. Blots are from three independent experiments. V) ELISA analysis of serum IgE levels in WT mice, asthma model mice, and asthma model mice treated with PGRN in vivo ( n = 6).

Article Snippet: Following incubation, the cells were fixed, permeabilized, and stained with a range of markers, including pCD19 (ab203615; Abcam), pBTK (5082S; Cell Signaling Technology), pY (05‐321; Merck‐Millipore), pSHIP (3941S; Cell Signaling Technology), and pWASP (A300‐205A; Bethyl Laboratories).

Techniques: Activation Assay, Gene Expression, Western Blot, Expressing, Control, Chromatin Immunoprecipitation, Purification, DNA Purification, Real-time Polymerase Chain Reaction, Luciferase, Reporter Assay, Activity Assay, Plasmid Preparation, In Vitro, Flow Cytometry, Labeling, Fluorescence, Enzyme-linked Immunosorbent Assay, In Vivo

Journal: Experimental and Therapeutic Medicine

Article Title: Interference of ALOX5 alleviates inflammation and fibrosis in high glucose‑induced renal mesangial cells

doi: 10.3892/etm.2022.11733

Figure Lengend Snippet: Silencing of ALOX5 alleviates inflammatory response and fibrosis in HG-induced SV40 MES-13 cells. (A-C) The HG-induced SV40 MES-13 cells were transfected with si-ALOX5 or si-control, and the inflammatory cytokines, including TNF-α, IL-6 and IL-8 were evaluated using their corresponding ELISA kits. (D) The expression level of fibrosis-related proteins was assessed using western blotting. *** P<0.001 vs. NG; ## P<0.01 and ### P<0.001 vs. HG + si-control. ALOX5, 5-lipoxygenase; HG, high glucose; si-, siRNA; ELISA, enzyme-linked immunosorbent; NG, normal glucose; COL4, collagen type IV; FN, fibronectin; TGF-β1, transforming growth factor β1.

Article Snippet: The concentrations of inflammatory cytokines in the culture medium, including TNF-α, IL-6 and IL-1β, were detected using their corresponding ELISA kits: Mouse TNF-α ELISA kit (cat. no. orb565170), mouse IL-6 ELISA kit (cat. no. orb565132) or mouse IL-8 ELISA kit (cat. no. orb566249) from Biorbyt Ltd., according to the manufacturer's instructions.

Techniques: Transfection, Control, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot

Journal: Journal of neuroinflammation

Article Title: Bruton's tyrosine kinase inhibition ameliorated neuroinflammation during chronic white matter ischemia.

doi: 10.1186/s12974-024-03187-4

Figure Lengend Snippet: Fig. 1 Ischemic demyelination and activation of BTK in microglia induced by BCAS. Ischemic demyelination and activation of BTK in microglia induced by BCAS. (A) The degree of white matter damage and the relative intensity of MBP in the CC at different time points after BCAS. Scale bar = 200 μm. (B) Sum marized data display the severity of CC myelin damage. One-way ANOVA and Tukey post hoc test, n = 6 mice/ group (biological replicates). (C) Proportions of various immune cells in BCAS 1 m brains. (D) Representative images of microglia immunofluorescence, skeletonized microglia, and the expression of BTK and p-BTK in microglia at several time points post BCAS. Scale bar = 10 μm. (E) Quantification analysis of the number, soma area, solidity, round of microglia and the percentage of BTK+microglia and p-BTK+microglia. One-way ANOVA and Tukey post hoc test, n = 6 mice/ group (biological replicates)

Article Snippet: Briefly, the concentration of extracted protein was quantified using BCA Protein Assay Kit (Promoter), then transferred to Nitrocellulose (NC, Satrorius) membranes and blocked with 5% BSA for 1 h, followed by incubated with primary antibodies including BTK (1:1000, 8647, CST) and pBTK (1:1000, NBP178295, NOVUS) overnight at 4 °C.

Techniques: Activation Assay, Immunofluorescence, Expressing

Journal: Pharmacology Research & Perspectives

Article Title: ZYBT1, a potent, irreversible Bruton’s Tyrosine Kinase (BTK) inhibitor that inhibits the C481S BTK with profound efficacy against arthritis and cancer

doi: 10.1002/prp2.565

Figure Lengend Snippet: ZTBT1 inhibited the phosphorylation of (A) BTK (Y223) and (B) PLCγ2 (Y1217). The IC 50 values are summarized in the following table. (C) ZYBT1 could also suppress the secretion of TNFα (filled circle), IL‐6 (square), and IL‐8 (open circle) in a cell based assay. The IC 50 values are shown the attached table. The data presented are representative of three independent experiments

Article Snippet: Human phospho‐BTK (Y223) antibody and human BTK antibody were from R&D system.

Techniques: Phospho-proteomics, Cell Based Assay

Journal: PeerJ

Article Title: Identification and validation of immune related core transcription factors GTF2I in NAFLD

doi: 10.7717/peerj.13735

Figure Lengend Snippet: The primer sequences.

Article Snippet: After protein quantification using a microplate reader, an appropriate amount of loading buffer was added and subsequently changed by heating in 95 °C for 15 min. After polyacrylamide gel electrophoresis (30 g protein usage) and subsequent routine western blot experimental steps, GAPDH (diluted 1:25,000, 60004-1, Proteintech, USA), CHD2 (diluted 1:1,000, 12311-1-AP, Proteintech, USA) were respectively incubated overnight with the corresponding PVDF membrane region with the GTF2I (1:1,000, ab248269, abcam, USA) antibody at 4 °C overnight.

Techniques:

Journal: PeerJ

Article Title: Identification and validation of immune related core transcription factors GTF2I in NAFLD

doi: 10.7717/peerj.13735

Figure Lengend Snippet: (A–B) The mRNA and protein of GTF2I was high expression in the FFA group, while the difference in CHD2 was not obvious. (C) H & E staining was used as pathological evidence for the diagnosis of NAFLD. (D–E) RT-qPCR revealed the mRNA expression of both GTF2I and CHD2 were significantly higher in NAFLD patients than in normal participants. (F) WB showed that GTF2I but not CHD2 was significantly higher in NAFLD patients than in normal participants. (G) The immunohistochemical results of GTF2I in liver tissues of NAFLD and normal participants. Arrows served as a marker of positive areas. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: After protein quantification using a microplate reader, an appropriate amount of loading buffer was added and subsequently changed by heating in 95 °C for 15 min. After polyacrylamide gel electrophoresis (30 g protein usage) and subsequent routine western blot experimental steps, GAPDH (diluted 1:25,000, 60004-1, Proteintech, USA), CHD2 (diluted 1:1,000, 12311-1-AP, Proteintech, USA) were respectively incubated overnight with the corresponding PVDF membrane region with the GTF2I (1:1,000, ab248269, abcam, USA) antibody at 4 °C overnight.

Techniques: Expressing, Staining, Biomarker Discovery, Quantitative RT-PCR, Immunohistochemical staining, Marker

Journal: Journal of Virology

Article Title: Ibrutinib inhibits the replication of multiple poxviruses by targeting the Bruton tyrosine kinase

doi: 10.1128/jvi.00517-25

Figure Lengend Snippet: Impact of BTK knockdown and overexpression on VACV-WR and MPXV infection. ( A ) HeLa cells were transfected with BTK siRNA (80 nM) and negative control siRNA for 48 h, then were infected with VACV-WR at 0.01 pfu/cell for 48 h. Samples were lysed and proteins resolved by SDS-PAGE and western blotting with anti-BTK, anti-VACV antiserum, and anti-GAPDH antibodies. VACV: anti-VACV antiserum. ( B ) HeLa cells were transfected with BTK siRNA (80 nM) and negative control siRNA for 48 h, then were infected with VACV-WR at 0.01 pfu/cell for 48 h. The whole cell lysates were collected and titered in BS-C-1 cells. ( C ) A549 cells were transfected with BTK siRNA (80 nM) and negative control siRNA for 48 h, then were infected with VACV-WR at 0.01 pfu/cell for 48 h. The whole cell lysates were collected and proteins were resolved by SDS-PAGE and western blotting with anti-BTK, anti-VACV antiserum, and anti-GAPDH antibodies. VACV: anti-VACV antiserum. ( D ) A549 cells were transfected with siBTK (80 nM) and negative control siRNA for 48 h, then were infected with VACV-WR at 0.01 pfu/cell. The whole cell lysate was collected at 48 hpi, and viruses within were titered in BS-C-1 cells. ( E ) HFF cells were transfected with BTK siRNA (80 nM) and negative control siRNA for 48 h, then were infected with VACV-WR at 0.01 pfu/cell for 48 h. Samples were lysed and proteins resolved by SDS-PAGE and western blotting with anti-BTK, anti-VACV antiserum, and anti-GAPDH antibodies. VACV: anti-VACV antiserum. ( F ) HFF cells were transfected with BTK siRNA (80 nM) and negative control siRNA for 48 h, then were infected with VACV-WR at 0.01 pfu/cell for 48 h. Viruses in whole cell lysate were collected and titered in BS-C-1 cells. ( G ) BTK-KO cells (2#,10#) were generated by CRISPR-Cas9 technology. ( H ) BTK-KO cells were infected with 3 pfu/cell WR for 24 h, and then the whole cell lysates were collected and viruses within were titered in BS-C-1 cells. ( I ) HeLa cells were transfected with BTK plasmids at 0.1, 0.5, and 1 µg for 24 h, then were infected with VACV-WR at 0.01 pfu/cell for 48 h. The whole cell lysates were collected, and proteins were lysed and resolved by SDS-PAGE and western blotting with anti-BTK, anti-VACV antiserum, and anti-GAPDH antibodies. VACV: anti-VACV antiserum. ( J ) HeLa cells were transfected with BTK plasmids at 0.1, 0.5, and 1 µg for 24 h, then were infected with VACV-WR at 0.01 pfu/cell. The whole cell lysates were collected at 48 hpi and viruses within were titered in BS-C-1 cells. NC: negative control. ( K ) HFF cells were transfected with BTK plasmids at 0.1, 0.5, and 1 µg for 24 h, then were infected with VACV-WR at 0.01 pfu/cell for 48 h. Samples were lysed and proteins resolved by SDS-PAGE and western blotting with anti-BTK, anti-VACV antiserum, and anti-GAPDH antibodies. VACV: anti-VACV antiserum. ( L ) HFF cells were transfected with BTK plasmids at 0.1, 0.5, and 1 µg for 24 h, then were infected with VACV-WR at 0.01 pfu/cell. Viruses in whole cell lysate were collected at 48 hpi and titered in BS-C-1 cells. NC: negative control. ( M ) A549 cells were transfected with BTK plasmid at 0.1, 0.5, and 1 µg for 24 h, then were infected with VACV-WR at 0.01 pfu/cell for 48 h. The whole cell lysates were collected, and proteins were lysed and resolved by SDS-PAGE and western blotting with anti-BTK, anti-VACV antiserum, and anti-GAPDH antibodies. VACV: anti-VACV antiserum. ( N ) A549 cells were transfected with BTK plasmids at 0.1, 0.5, and 1 µg for 24 h, then were infected with VACV-WR at 0.01 pfu/cell. The whole cell lysates were collected at 48 hpi and viruses within were titered in BS-C-1 cells. NC: negative control. ( O ) HFF cells were transfected with BTK siRNA or BTK plasmids at 0.1, 0.5, and 1 µg for 48 h. Samples were lysed and proteins resolved by SDS-PAGE and western blotting with anti-BTK antibody. ( P and Q ) HeLa cells were transfected with BTK siRNA (80 nM) and negative control siRNA, and then the cells were infected with MPXV at 3 pfu/cell. At 24 hpi, the expression of viral proteins in infected cells was detected by western blotting using the indicated antibodies (Fig. 5P), and virus titers were determined by a plaque assay (Fig. 5Q). Data ( A–F, H–N, P and Q ) were collected from three independent experiments, and they are presented as mean ± standard deviation. One-way ANOVA followed by Tukey’s multiple comparisons test was used to analyze the data. Statistics: n.s., not significant; P > 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: BTK and its mutants (Y223F, C481Y, Y551F) plasmids were constructed by our lab. Phospho-Btk (Tyr223) Antibody (#5082) was purchased from Cell Signaling Technology; BTK Antibody (CSB-PA002867LA01HU) was purchased from CUSABIO; Rabbit antibody to VACV-WR strain and mouse antibodies to VACV-I3 or VACV-E3 were described previously ( , ); mouse anti-A3 and LSDV antibodies were prepared by our lab; mouse anti-GAPDH antibody (#AF0006), mouse anti-histone H3 antibody (#AF0009), mouse anti-HA antibody (#AH158), HRP-labeled goat anti-mouse IgG (#A0216), and HRP-labeled goat anti-rabbit IgG (#A0208) were purchased from Beyotime Biotechnology; Goat anti-rabbit IgG Alexa Fluor 555 (#A32732) was purchased from Thermo Fisher Scientific.

Techniques: Knockdown, Over Expression, Infection, Transfection, Negative Control, SDS Page, Western Blot, Generated, CRISPR, Plasmid Preparation, Expressing, Virus, Plaque Assay, Standard Deviation

Journal: Journal of Virology

Article Title: Ibrutinib inhibits the replication of multiple poxviruses by targeting the Bruton tyrosine kinase

doi: 10.1128/jvi.00517-25

Figure Lengend Snippet: BTK was phosphorylated upon VACV-WR infection, and point mutations of BTK exhibited decreased ability to promote viral replication. ( A ) HeLa cell lines were either uninfected or infected with VACV-WR at 3 pfu/cell in the presence or absence of ibrutinib, and samples were harvested at the specified time. Phosphorylation levels of BTK protein and total BTK protein levels were determined via western blotting. ( B ) The ratio of phosphorylated BTK to total BTK protein level was measured and calculated by ImageJ. ( C ) Schematic diagram of BTK and its mutants. ( D ) HeLa-BTK-KO cells in triplicate were transfected with BTK and BTK mutants for 24 h, and then were infected with 3 pfu/cell WR for 6 h, and samples were collected. E11L was detected by qPCR to detect the effect of mutants on DNA levels. NC: negative control ( E ) BTK and BTK mutants were transfected into HeLa-BTK-KO cells in triplicate for 24 h, and the cells were infected with 0.01 pfu/cell WR, and viruses were collected at 48 hpi and titered in BS-C-1 cells. NC: negative control ( F ) BTK and BTK mutants were transfected into HeLa-BTK-KO cells for 24 h, and then the cells were infected with 0.01 pfu/cell WR for 24 h. The expression of viral proteins was detected by western blotting analysis after the samples were collected. NC, negative control; VACV, anti-VACV antiserum.

Article Snippet: BTK and its mutants (Y223F, C481Y, Y551F) plasmids were constructed by our lab. Phospho-Btk (Tyr223) Antibody (#5082) was purchased from Cell Signaling Technology; BTK Antibody (CSB-PA002867LA01HU) was purchased from CUSABIO; Rabbit antibody to VACV-WR strain and mouse antibodies to VACV-I3 or VACV-E3 were described previously ( , ); mouse anti-A3 and LSDV antibodies were prepared by our lab; mouse anti-GAPDH antibody (#AF0006), mouse anti-histone H3 antibody (#AF0009), mouse anti-HA antibody (#AH158), HRP-labeled goat anti-mouse IgG (#A0216), and HRP-labeled goat anti-rabbit IgG (#A0208) were purchased from Beyotime Biotechnology; Goat anti-rabbit IgG Alexa Fluor 555 (#A32732) was purchased from Thermo Fisher Scientific.

Techniques: Infection, Phospho-proteomics, Western Blot, Transfection, Negative Control, Expressing

Journal: Journal of Virology

Article Title: Ibrutinib inhibits the replication of multiple poxviruses by targeting the Bruton tyrosine kinase

doi: 10.1128/jvi.00517-25

Figure Lengend Snippet: During virus infection, BTK can enter the nucleus. ( A ) HeLa and ( B ) THP-1 cells were either untreated or infected with VACV at 3 pfu/cell for the indicated time in the presence or absence of ibrutinib (10 µM). A confocal experiment was performed to detect the subcellular localization of BTK. ( C ) The confocal analyses were performed in triplicate, and the localization of BTK was quantified in 50 randomly selected HeLa cells, and bars represent mean values ± SD. ( D ) The confocal analyses were performed in triplicate, and the localization of BTK was quantified in 50 randomly selected THP-1 cells, and bars represent mean values ± SD. ( E and F ) HeLa cells were infected with VACV-WR at 3 pfu/cell for the indicated time in the presence or absence of ibrutinib (10 µM), and then nuclear and cytoplasmic proteins were extracted using a nucleoplasmic separation kit and subjected to western blotting analysis with anti-BTK, anti-VACV antiserum, anti-histone H3 (H3 stands for Histone H3, a housekeeping gene of the nucleus), and anti-GAPDH antibodies. VACV: anti-VACV antiserum. ( G ) HeLa cells were infected with VACV-WR for 2 h and then were transfected with BTK and its mutants (WT, Y551F, and Y223F) for 24 h. After that, a confocal experiment was performed to detect the subcellular localization of BTK and its mutants. ( H ) The confocal microscopic analysis was performed in triplicate, and the localization of BTK was quantified in 50 randomly selected HeLa cells, and bars represent mean values ± SD.

Article Snippet: BTK and its mutants (Y223F, C481Y, Y551F) plasmids were constructed by our lab. Phospho-Btk (Tyr223) Antibody (#5082) was purchased from Cell Signaling Technology; BTK Antibody (CSB-PA002867LA01HU) was purchased from CUSABIO; Rabbit antibody to VACV-WR strain and mouse antibodies to VACV-I3 or VACV-E3 were described previously ( , ); mouse anti-A3 and LSDV antibodies were prepared by our lab; mouse anti-GAPDH antibody (#AF0006), mouse anti-histone H3 antibody (#AF0009), mouse anti-HA antibody (#AH158), HRP-labeled goat anti-mouse IgG (#A0216), and HRP-labeled goat anti-rabbit IgG (#A0208) were purchased from Beyotime Biotechnology; Goat anti-rabbit IgG Alexa Fluor 555 (#A32732) was purchased from Thermo Fisher Scientific.

Techniques: Virus, Infection, Western Blot, Transfection