ifitm3 inhibitor caraphenol a  (MedChemExpress)

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: The B cells from the WT and PGRN KO mice were isolated for in vitro treatment with the IFITM3 inhibitor caraphenol A (30 μ m ; HY‐N3540, MCE) or DMSO.

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