Fig 1: Validation of increased CXCR2high cells in the callus of aged mice with increased IRF and NF-?B pathways and reduced osteogenic potential. The tibial fracture was performed on 4-month- (young) and 21-month-old (aged) C57BL/6J male mice and fracture callus was harvested 10 days after the procedure. (A) Immunostaining with anti-CXCR2 antibody in callus sections for CXCR2+ cells. Images show numerous CXCR2+ cells (arrows) localize on the surface of woven bone. The percentage of CXCR2+ cells in the callus of young and aged mice was quantified by Image J software. n = 4 mice/young and 3 mice/aged. Data represent mean ± SD. Unpaired two-tailed t-test. *p < 0.05. WB: Woven bone. Scale bar = 1 mm. (B) Western blot to assess IRF-response protein IFITM1 and NF-?B-response protein S100A6 in young and aged callus tissues. (C) Violin plot of the expression level of Cxcr2 in cluster 1.1 and cluster 1.2. (D) CXCR2low and CXCR2high cells were isolated from callus-derived mesenchymal progenitor culture. (E) The expression levels of target genes for IRF and NF-?B were measured by RT-qPCR. n = 3 mice/group. Data represent mean ± SD. Unpaired two-tailed t-test. *p < 0.05. (F) CXCR2low and CXCR2high cells isolated from callus-derived mesenchymal progenitor culture were cultured in osteoblast inducing medium for 7 days and stained for ALP. The percentage area of ALP+ was measured in Image J software. Expression level of Alp and Runx2 was measured with RT-qPCR. Data represent mean ± SD. Unpaired two-tailed t-test. *p < 0.05.