Journal: Cell Communication and Signaling : CCS

Article Title: Neutrophil membrane-coated circular RNA nanoparticles for targeted immunotherapy in HER2-positive breast cancer brain metastasis

doi: 10.1186/s12964-025-02321-w

Figure Lengend Snippet: Construction and Characterization of HER2-Targeted dHL-60 Cell Membranes. (A) Schematic representation of the lentiviral vector design used to overexpress the scFv targeting HER2. The vector includes the anti-HER2 scFv, a membrane localization signal, a FLAG tag for detection, a hinge region for flexibility, and a transmembrane domain to anchor the scFv to the cell membrane. (B) SPR analysis illustrating the binding affinity of scFv to the HER2 protein. (C) Flow cytometry analysis comparing the binding of scFv (anti-HER2) to SKBR3 cells versus a control scFv. (D) Flow cytometry assessment of CD11b expression levels in HL-60 cells before and after differentiation induction with DMSO. (E) Western blot results demonstrating the expression of adhesion molecules CD11b, CD18, CD49d, and CD31 in differentiated HL-60 cells. (F) Western blot analysis was performed using an anti-FLAG antibody to detect the FLAG-tagged scFv (anti-HER2). A band corresponding to the scFv-FLAG fusion protein is observed in dHL-60 cells transduced with the lentiviral vector, but not in control cells. (G) Flow cytometry analysis (anti-FLAG antibody) demonstrating the surface expression of scFv (anti-HER2) on dHL-60 cells overexpressing scFv (anti-HER2). (H) Illustration depicting the construction and extraction process of the cell membrane from HL-60 cells, outlining the steps involved in preparing membrane-coated nanoparticles

Article Snippet: CoraLite ® Plus 488, PE-conjugated Goat Anti-Rabbit IgG, antibodies against CD11B, CD49d, CD31, CD18, β-actin, His Tag, FLAG Tag and CXCL9 were obtained from Proteintech (Wuhan, China).

Techniques: Plasmid Preparation, Membrane, FLAG-tag, Binding Assay, Flow Cytometry, Control, Expressing, Western Blot, Transduction, Extraction

Journal: The Journal of Neuroscience

Article Title: Suppression of Inflammation with Conditional Deletion of the Prostaglandin E 2 EP2 Receptor in Macrophages and Brain Microglia

doi: 10.1523/JNEUROSCI.2203-13.2013

Figure Lengend Snippet: The EP2 receptor induces expression of inflammatory enzymes and cytokines in mouse peritoneal macrophages. For all panels: *p < 0.05, **p < 0.01, ***p < 0.001, values are mean ± SEM. A, EP2 immunostaining is detected in wild-type but not EP2−/− C57BL/6 primary peritoneal macrophages costained for Cd11b. Scale bar, 100 μm. B, Peritoneal macrophages were stimulated with LPS (10 ng/ml) for 1 and 6 h. qPCR demonstrates a rapid upregulation of EP2 receptor mRNA by 1 h and subsequent downregulation by 6 h following LPS stimulation (n = 4–6 per group; two-way ANOVA for effect of time ##p < 0.01, and effect of interaction p = 0.02; Bonferroni's multiple-comparison tests comparing mean of 1 h vehicle (veh) and 1 h LPS *p < 0.05). C, Peritoneal macrophages were stimulated with LPS (10 ng/ml) +/− the EP2 agonist butaprost (1 μm) or vehicle. qPCR demonstrates an induction of proinflammatory mediators COX-2, iNOS, and gp91phox with LPS stimulation that is further enhanced by costimulation with butaprost (time points 1 h for COX-2 and 6 h for iNOS and gp91phox; n = 4–6 per group; two-way ANOVA for effect of LPS #p < 0.05, ###p < 0.001; Bonferroni's multiple-comparison tests comparing means of LPS-con and LPS-butaprost *p < 0.05, **p < 0.01). D, LPS-induced macrophage NO release is increased by EP2 receptor activation with butaprost (1 μm), whereas EP2−/− macrophages show reduced NO levels compared with EP2+/+ macrophages (n = 4 per group; Student's tests #p < 0.05, ##p < 0.01 comparing EP2+/+ to EP2−/−). E, qPCR demonstrates induction of IL-6 mRNA at 1 h and IL1β mRNA at 6 h, and a trend of decreased TNFα mRNA at 6 h in LPS-stimulated macrophages with addition of butaprost (n = 4–6 per group; two-way ANOVA for effect of LPS ##p < 0.01, ###p < 0.001; Bonferroni's multiple-comparison tests comparing means of LPS-con and LPS-butaprost **p < 0.01 for IL-6 at 1 h and IL1β at 6 h).

Article Snippet: Cells were purified with anti-mouse Cd11b Ab-conjugated magnetic beads and MACS columns (Miltenyi Biotec), as previously described ( Shi et al., 2010 ).

Techniques: Expressing, Immunostaining, Comparison, Activation Assay

Journal: The Journal of Neuroscience

Article Title: Suppression of Inflammation with Conditional Deletion of the Prostaglandin E 2 EP2 Receptor in Macrophages and Brain Microglia

doi: 10.1523/JNEUROSCI.2203-13.2013

Figure Lengend Snippet: Conditional deletion of the EP2 receptor in macrophages suppresses oxidative enzyme and cytokine gene expression. A, Genomic DNA PCR is shown for EP2+/+, EP2lox/+, and EP2lox/lox C57BL/6 mice. B, Quantitative genomic PCR was assayed for EP2+/+ wild-type, EP2+/−, EP2−/−, Cd11bCre;EP2lox/lox, Cd11bCre;EP2lox/+, and Cd11bCre;EP2+/+; values are relative to wild-type EP2+/+ control DNA. C, Peritoneal macrophages were isolated from adult Cd11bCre;EP2lox/lox and Cd11bCre;EP2+/+ mice and sorted using Cd11b antibody-conjugated magnetic beads before qPCR analysis. Basal levels of EP2 mRNA, assayed by qPCR, are reduced by 91% in Cd11bCre;EP2lox/lox compared with control macrophages (left; Cd11bCre;EP2lox/lox vs Cd11bCre;EP2+/+) and are reduced by 56% in LPS-stimulated Cd11bCre;EP2lox/lox macrophages (right; **p < 0.01; n = 5–6 per group). D, Conditional deletion of EP2 in macrophages reduces LPS-mediated increases in proinflammatory gene expression (two-way ANOVA for effect of LPS treatment is represented by ###p < 0. 001; Bonferroni's multiple-comparisons tests comparing mean of Cd11bCre;EP2+/+/LPS and Cd11bCre;EP2lox/lox/LPS were ***p < 0.001; n = 5–6 per group).

Article Snippet: Cells were purified with anti-mouse Cd11b Ab-conjugated magnetic beads and MACS columns (Miltenyi Biotec), as previously described ( Shi et al., 2010 ).

Techniques: Gene Expression, Control, Isolation, Magnetic Beads

Journal: The Journal of Neuroscience

Article Title: Suppression of Inflammation with Conditional Deletion of the Prostaglandin E 2 EP2 Receptor in Macrophages and Brain Microglia

doi: 10.1523/JNEUROSCI.2203-13.2013

Figure Lengend Snippet: Examination of levels of monocytic populations in Cd11bCre;EP2+/+ and Cd11bCre;EP2lox/lox mice. Splenocytes and peripheral blood immune cells were isolated from 3-month-old mice. A, Representative plots for CD11b+ cells gated on CD115 and Ly6C yielding four populations of cells, including CD115−/Ly6C− macrophages, CD115−/Ly6Cint-hi neutrophils, CD115int/Ly6Cint resident monocytes, and CD115hi-int/Ly6Chi inflammatory monocytes in vehicle and LPS-treated mice. B, Quantification of levels of monocytic populations, including macrophages, resident monocytes, and inflammatory monocytes does not show differences between genotypes in vehicle or LPS-treated mice. Levels of neutrophils are decreased in peripheral blood with LPS, but not vehicle stimulation (*p < 0.05; n = 4 mice per group). C, Quantification of CD11b-positive microglia derived from brains of Cd11bCre;EP2+/+ and Cd11bCre;EP2lox/lox mice does not show differences in number (n = 5–7 mice per genotype). D, Comparison of copy number of EP2/copy number of 18S is shown for adult microglia and peritoneal macrophages from Cd11bCre;EP2+/+ and Cd11bCre;EP2lox/lox mice (n = 4–6 per group; p < 0.05 unpaired t test). Macrophage expression of EP2 in Cd11bCre;EP2+/+ mice was 28-fold higher; however, the percentage reduction of expression with conditional deletion of EP2 was similar in both microglia and macrophages, and was 62.2 and 62.1%, respectively.

Article Snippet: Cells were purified with anti-mouse Cd11b Ab-conjugated magnetic beads and MACS columns (Miltenyi Biotec), as previously described ( Shi et al., 2010 ).

Techniques: Isolation, Derivative Assay, Comparison, Expressing