Extended Data Fig. 5. CGRP expression in neutrophils and macrophages, its effects on Ramp1–/– cells, M2-Like macrophages, Ly6C expression, and CD11b+ cell apoptosis.
a, Calcrl and Ramp1 expression in bone marrow-derived neutrophils and macrophages detected by RT-PCR. Gapdh was used as the housekeeping gene. Repeated independently 3 times. For gel source data, see Supplementary Fig. 1. b, CALCRL and RAMP1 expression was detected in bone marrow-derived neutrophils and macrophages using immunostaining. CALCRL, green; RAMP1, red; nuclei, blue. Scale bars = 25 μm. Repeated independently 3 times. c, Neutrophils and macrophages derived from mouse bone marrow of LysMCre+/–/Ramp1fl/fl were treated with saline (PBS, 0 nM CGRP) or CGRP (1 nM) and transwell migration towards a chemoattractant (CXCL1 or CCL2) was tested (n = 4). Results are expressed as fold change over the saline PBS/0 nM CGRP control group. d,e, Bone marrow-derived macrophages were treated with saline (0 nM CGRP) or CGRP (1 or 20 nM). Cell death in response to CGRP when macrophages were cultured with anti-inflammatory cytokines (IL-4/IL-13, or IL-10) (d) (n = 6). Ly6C expression in response to CGRP treatment after macrophage culture in inflammatory (TNF/IL-1) or anti-inflammatory conditions (IL-10) (e) (n = 6). Results are expressed as fold increase over treatment without CGRP and without cytokines. MFI is the geometric-mean of fluorescence intensity. f, Representative images of TUNEL assay at D3 post-injury in Rosa26DTA and Nav1.8Cre/Rosa26DTA mice (CD11b, green; TUNEL, red; nuclei, blue). Scale bar = 100 μm. Repeated independently 6 times. All data are plotted in box plots showing median (central line) and IQR (bounds). Whiskers show min. to max. range. Dots represent independent experiments. Two-way ANOVA with Bonferroni post hoc test for pair-wise comparisons. n.s., non-significant.