Journal: The Journal of Biological Chemistry

Article Title: Calcitonin gene-related peptide inhibits macrophage migration and differentiation via the GTPase Rap1

doi: 10.1016/j.jbc.2025.110949

Figure Lengend Snippet: BIBN alleviates CGRP-induced inhibition of macrophage migration. A, representative images of the scratch assay showing macrophage migration in the control, CGRP, BIBN, and BIBN + CGRP groups at 0 h and 24 h. The red dashed lines indicate the wound edges. This scale bar represents = 500 μm. Quantification of scratch (n = 3/group) on the right . B, representative images of the Transwell migration assay showing migrated macrophages in the control, CGRP, BIBN, and BIBN + CGRP groups. Cells that migrated through the membrane were stained with crystal violet . This scale bar represents = 100 μm. Quantification of Transwell assays (n = 7/group) on the right . C, representative images of phalloidin staining of BMMs cultured in the control, CGRP, BIBN, and BIBN + CGRP groups for 24 h, The scale bar represents = 20 μm; shape factor(n = 6/group)and length(n = 7/group). D, representative images showing F-actin ( red ) and Vinculin ( green ) staining of BMMs cultured in control, CGRP, BIBN, and BIBN + CGRP groups for 24 h, This scale bar represents = 10 μm. This scale bar represents in zoom = 1.5 μm; white arrows indicate filopodia (F-actin) and adhesion sites (Vinculin). E, quantification of filopodia and cell adhesion site is shown on the right . n = 6/group. F, expression of Rap1, PI3k, p-PI3k, Akt, p-Akt was examined by Western blot. β-actin was used as a loading control. Quantitative analyses of the relative band intensities of Rap1 to β-actin and p-AKTto AKT (n = 3). The p values were calculated by one-way analysis of variance. All data are presented as mean ± SD.

Article Snippet: To further investigate the effects of CGRP treatments on signaling pathways, 5 μm PI3K/AKT-IN-1 (Cat#HY-144806, MedChem-Express) was used to inhibit the PI3K-Akt signaling pathway.

Techniques: Inhibition, Migration, Wound Healing Assay, Control, Transwell Migration Assay, Membrane, Staining, Cell Culture, Expressing, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Calcitonin gene-related peptide inhibits macrophage migration and differentiation via the GTPase Rap1

doi: 10.1016/j.jbc.2025.110949

Figure Lengend Snippet: siRNA depletion of Rap1 reversed CGRP-induced phenotypes in macrophages. A, representative images of the scratch assay showing macrophage migration in the negative control (NC), negative control + CGRP, sirap1and sirap1+CGRP groups at 0 h and 24 h. The red dashed lines indicate the wound edges. This scale bar represents = 500 μm. Quantification of scratch (n = 3/group) on the right . B, representative images of the Transwell migration assay showing migrated macrophages in the negative control, negative control + CGRP, sirap1and sirap1+CGRP groups. Cells that migrated through the membrane were stained with crystal violet . This scale bar represents = 100 μm. Quantification of Transwell assays (n = 7/group) on the right . C, representative images of phalloidin staining of BMMs cultured in media containing negative control, negative control + CGRP, sirap1and sirap1+CGRP for 24 h, this scale bar represents = 20 μm; shape factor and length. n = 7. D, representative images showing F-actin ( red ) and Vinculin ( green ) staining of BMMs. This scale bar represent = 10 μm. This scale bar represents in zoom =1.5 μm. White arrows indicate filopodia (F-actin) and adhesion sites (Vinculin). E, quantification of filopodia and cell adhesion sites. n = 7/group. F, expression of E-cadherin, Vimentin, N-cadherin, PI3K, p-PI3K, p-AKT, AKT was examined by Western blot, GAPDH was used as a loading control. G, quantitative analyses of the relative band intensities of E-cadherin, N-cadherin, and Vimentin to GAPDH and p-PI3K to PI3K, p-AKT to AKT (n = 3). The p values were calculated by one-way analysis of variance ( A , B , C , and E ) or two-way analysis of variance ( G ). All data are presented as mean ± SD. CGRP, calcitonin gene-related peptide.

Article Snippet: To further investigate the effects of CGRP treatments on signaling pathways, 5 μm PI3K/AKT-IN-1 (Cat#HY-144806, MedChem-Express) was used to inhibit the PI3K-Akt signaling pathway.

Techniques: Wound Healing Assay, Migration, Negative Control, Transwell Migration Assay, Membrane, Staining, Cell Culture, Expressing, Western Blot, Control

Journal: The Journal of Biological Chemistry

Article Title: Calcitonin gene-related peptide inhibits macrophage migration and differentiation via the GTPase Rap1

doi: 10.1016/j.jbc.2025.110949

Figure Lengend Snippet: The PI3K/AKT inhibitor further suppressed macrophage migration. A, representative images of the scratch assay showing macrophage migration in the control, CGRP, PI3K/AKT-IN-1, PI3K/AKT-IN-1+CGRP groups at 0 h and 24 h. The red dashed lines indicate the wound edges. The scale bar represnts = 500 μm. Quantification of scratch (n = 3/group) on the right . B, representative images of the Transwell migration assay showing migrated macrophages in the control, CGRP, PI3K/AKT-IN-1, PI3K/AKT-IN-1+CGRP groups. Cells that migrated through the membrane were stained with crystal violet . Scale bar = 100 μm. Quantification of Transwell assays(n = 8/group) on the right . C, representative images of phalloidin staining of BMMs cultured in control, CGRP, PI3K/AKT-IN-1, PI3K/AKT-IN-1+CGRP groups for 24 h, This scale bar represent= 20 μm; shape factor, and length (n = 8). D , representative images showing F-actin ( red ) and Vinculin ( green ) staining of BMMs cultured in control, CGRP, PI3K/AKT-IN-1, PI3K/AKT-IN-1+CGRP groups for 24 h, This scale bar represents = 10 μm. This scale bar represents in zoom =1.5 μm. White arrow s indicate filopodia (F-actin) and adhesion sites (Vinculin). Quantification of filopodia and cell adhesion site is shown on the right . n = 8. The p values were calculated by one-way analysis of variance. All data are presented as mean ± SD. CGRP, calcitonin gene-related peptide.

Article Snippet: To further investigate the effects of CGRP treatments on signaling pathways, 5 μm PI3K/AKT-IN-1 (Cat#HY-144806, MedChem-Express) was used to inhibit the PI3K-Akt signaling pathway.

Techniques: Migration, Wound Healing Assay, Control, Transwell Migration Assay, Membrane, Staining, Cell Culture

Journal: BMC Immunology

Article Title: TRPV1 + neurons alter Staphylococcus aureus skin infection outcomes by affecting macrophage polarization and neutrophil recruitment

doi: 10.1186/s12865-023-00584-x

Figure Lengend Snippet: S. aureus activates cutaneous TRPV1 + neurons to release CGRP. a DRG was isolated from WT mice and stained with DAPI (blue), TRPV1 (green), and CGRP (red); scale bars, 200 μm. b Primary DRG neurons isolated from 6-week-old WT mice were stained with DAPI (blue), TRPV1 (green), and CGRP (red); white arrows show colocalization of TRPV1 with CGRP; scale bars, 50 μm. c CGRP release from dorsal skin tissue of TRPV1 −/− and WT mice before and 8 h after infection ( n = 4/group); One-way ANOVA with Tukey’s post hoc test. d Pretreatment of mice with RTX or PBS and CGRP release from dorsal skin tissue before and 8 h after infection ( n = 4/group); One-way ANOVA with Tukey’s post hoc test. e DRG neurons were stimulated with different concentrations of S. aureus supernatant or capsaicin for 30 min, and the release of CGRP was analyzed ( n = 4/group). One-way ANOVA with Tukey’s post hoc test. Data were pooled from two or three independent experiments

Article Snippet: Then, after treatment with CGRP (100 nM, GenScript, #RP11095) or vehicle for 2 h, M1 polarization was induced with IFN-γ (10 ng/ml, Novoprotein, #CM41), M2 polarization was induced with IL-4 (40 ng/ml, Novoprotein, #CK74), and subsequent experiments were performed 24 h later.

Techniques: Isolation, Staining, Infection

Journal: BMC Immunology

Article Title: TRPV1 + neurons alter Staphylococcus aureus skin infection outcomes by affecting macrophage polarization and neutrophil recruitment

doi: 10.1186/s12865-023-00584-x

Figure Lengend Snippet: CGRP regulates the polarization of BMDMs and the release of inflammatory factors. a - c BMDMs cultured in vitro were induced to M1 polarization with IFN-γ and M2 polarization with IL-4 The polarized macrophages were treated with CGRP or PBS and stained with DAPI (blue), CD80 (green), and CD206 (red); the ratio of CD80 + and CD206 + was analyzed under different intervention conditions. No difference in the confluency of BMDMs was observed among different groups. Scale bars, 20 μm. One-way ANOVA with Tukey’s post hoc test. d - f Expression levels of TNFα, IL-1β, and IL-10 in BMDMs after polarization in vitro under different experimental conditions; one-way ANOVA with Tukey posttests. Data were pooled from two or three independent experiments

Article Snippet: Then, after treatment with CGRP (100 nM, GenScript, #RP11095) or vehicle for 2 h, M1 polarization was induced with IFN-γ (10 ng/ml, Novoprotein, #CM41), M2 polarization was induced with IL-4 (40 ng/ml, Novoprotein, #CK74), and subsequent experiments were performed 24 h later.

Techniques: Cell Culture, In Vitro, Staining, Expressing

Journal: BMC Immunology

Article Title: TRPV1 + neurons alter Staphylococcus aureus skin infection outcomes by affecting macrophage polarization and neutrophil recruitment

doi: 10.1186/s12865-023-00584-x

Figure Lengend Snippet: Graphical abstract. In Staphylococcus aureus skin infection, local TRPV1 + neurons release CGRP to inhibit neutrophil recruitment and regulate macrophage polarization, resulting in the aggravation of local infection

Article Snippet: Then, after treatment with CGRP (100 nM, GenScript, #RP11095) or vehicle for 2 h, M1 polarization was induced with IFN-γ (10 ng/ml, Novoprotein, #CM41), M2 polarization was induced with IL-4 (40 ng/ml, Novoprotein, #CK74), and subsequent experiments were performed 24 h later.

Techniques: Infection