Macrophages expressing BRAFV600E recapitulate the pathologic phenotype of ECD. (A) Schematic representation of the lentiviral vector expressing BRAFV600E. (B) Experimental set up: monocytes isolated from the buffy coats of healthy donors were transduced with lentiviral constructs and cultured for 7 days before harvesting and analysis. (C) Expression level of wtBRAF (WT) and BRAFV600E (VE) as determined by GFP in flow cytometry; 10 independent donors are shown, with statistical significance evaluated with Friedman test with Dunn’s multiple-comparisons test; ns nonsignificant. (D) Flow cytometric analysis of forward and side scatter rates (FSC and SSC, respectively) of nontransduced macrophages (UT) or macrophages expressing wtBRAF (WT) and BRAFV600E (VE); 7 independent donors are shown, with statistical significance evaluated with Friedman test with Dunn’s multiple-comparisons test. ***P < .01. (E) Representative widefield images of staining with Oil Red O of nontransduced macrophages (UT) or macrophages expressing wtBRAF (WT) and BRAFV600E (VE). Cells were fixed, stained with DAPI and Oil Red O solution, and analyzed by widefield fluorescence microscopy to observe wtBRAF and BRAFV600E expression level (as determined by GFP detection), DAPI-stained nuclei, and Oil Red-O staining. Scale bars, 20 μm. (F) Oil Red-O staining of primary macrophages isolated from an ECD lesion (skin biopsy specimen, original magnification x400). Scale bar, 100 μm. (G) Immunohistochemistry staining showing phosphorylated ERK in macrophages infiltrating ECD lesions (skin biopsy specimen). (H) Western blot analysis of phosphorylated ERK1/2 (p-ERK1/2) and phosphorylated p38 (p-p38) in nontransduced macrophages (UT) or macrophages expressing wtBRAF (WT) and BRAFV600E (VE). The levels of total ERK1/2 and total p38 in whole-cell lysates were used as the protein loading control.