Classical autophagy markers are absent from PRR-induced Atg8/LC3-positive phagosomes. (A) Macrophages transduced with lentiviral constructs of fluorescent-tagged Atg8/LC3 were stained for the indicated proteins and DAPI. Arrows point to Atg8/LC3-positive phagosomes. Insets show a zoomed-in view of the phagosomes of interest. (B) Pearson’s coefficient values were calculated for each marker in Atg8/LC3-positive phagosomes (n ≥ 20). Mean values are represented as horizontal lines. A Pearson score >0.6 is indicated in each graph as a dashed line. poly-ub, polyubiquitin. (C) Zymosan-containing phagosomes in macrophages transduced with the tandem fluorescent-tagged Atg8/LC3 construct and treated with zymosan for 6 h. (D) Macrophages transduced with tandem fluorescent Atg8/LC3 and treated or not treated with concanamycin A (ConA). (E) Representative GFP immunogold labeling in electron micrographs of phagosomes harboring heat-killed C. albicans in GFP-Atg8/LC3–transduced macrophages. The areas highlighted by dashed line boxes are shown with higher magnification on the right. Arrows point to the single membrane structure labeled with gold particles.