The ferritin–NCOA4 condensates are targeted by both macroautophagy and endosomal microautophagy. (A) Ferritin–NCOA4 condensates are engulfed by autophagosomes. WT HeLa cells expressing mGFP-FTH1 (green) and Halo-LC3 (red) were treated with 50 μg/ml FAC for 24 h followed by 50 μM DFO for 5 h and then observed by time-lapse fluorescence microscopy at 2-min intervals (see also Video 1). Arrows indicate part of ferritin condensates engulfed by an autophagosome. Scale bars, 5 μm (main) and 1 μm (magnified). (B) 3D-CLEM of cells treated as in A. Arrowheads indicate the surface of a ferritin–NCOA4 condensate exposed to the cytosol, and arrows indicate an elongating autophagosomal membrane. Scale bar, 500 nm. (C) The ferritin–NCOA4 condensates are incorporated into endosomes. WT HeLa cells expressing GFP-FTH1 or mGFP-NCOA4 were treated with 2 μg/ml doxycycline for 48 h to induce mRuby3-RAB5^Q79L expression and then observed by time-lapse fluorescence microscopy at 4-s intervals under normal growing conditions (see also Videos 2, 3, and 4). Arrowheads indicate ferritin–NCOA4 condensates in the cytosol, and arrows indicate the ferritin–NCOA4 condensates in enlarged endosomes. Scale bars, 5 μm. (D and E) 3D-CLEM of WT HeLa cells expressing mGFP-FTH1 treated as in C. Correlative scanning electron microscopy (SEM) and fluorescent images are shown. An enlarged endosome containing mGFP-FTH1 puncta is magnified. Scale bars, 5 μm (main) and 1 μm (magnified; D). TEM images of the enlarged endosome indicated by arrows in SEM and fluorescent images are shown. Scale bars, 5 μm (SEM and fluorescent images), 1 μm (TEM), and 100 nm (magnified TEM; E). (F)NCOA4 KO HeLa cells expressing mGFP-FTH1 were examined as in C. Scale bar, 10 μm.