Fig. 7. Myeloid EGFR^−/− mice had decreased subpopulations of ATMs in diet-induced obesity.

A An efferocytosis assay was performed by ex vivo co-culturing BMDMs (labeled with Cytotell Blue) isolated from WT and myeloid EGFR^−/− mice with neutrophils isolated from WT mice that were treated with Staurosporine to induce apoptosis and labeled with CFSE. The first quadrant (in red) represents the efferocytosing monocyte population. Flow cytometry analysis showed that EGFR^−/− BMDMs had enhanced efferocytosis of apoptotic neutrophils, as indicated by a higher ratio of phagocytic to non-phagocytic cells. N = 4 and 5. B Flow cytometric analysis of epididymal fat ATMs (gating on CD45 + , Ly6G-, CD3-, CD4-, CD8-, SiglecF-) determined that both CD11b + , Ly6C + (Ly6C + ) ATMs and CD11b + , Ly6C-, CD64 + , F4/80 + , CD9 + (Ly6C-CD9 + ) ATMs were lower in MΦ EGFR^−/− mice than WT mice fed the HFD for 6 weeks. N = 5. C Double immunofluorescent staining determined that both CD9 and AREG expression was evident and colocalized in ATMs in CLSs in WT mice with HFD while their expression was lower in MΦ EGFR^−/− mice on HFD and minimal in both WT and MΦ EGFR^−/− mice on chow food. Scale bar = 100 μm. Data are means ± SEM, ***P < 0.001, analyzed using 2 tailed Student’s t test.