Cargo internalization into APPL endosomes is clathrin dependent but their biogenesis is not. (A) Silencing of CHC by RNAi in HeLa cells assessed by Western blot in comparison to EEA1, APPL1, and Rab5 as controls. (B) Internalization of biotinylated Tf (b-Tf) (after 30 min of continuous uptake) is inhibited upon CHC knockdown. The amounts of b-Tf in cell lysates were quantified by electrochemiluminescence. (C) Knockdown of CHC decreased colocalization of Tf to EEA1 (red) and APPL1 (blue). Colocalization was quantified after 3.5-min chase after 0.5-min internalization pulse of Tf. (D–F) Knockdown of clathrin inhibits Tf uptake but does not affect the number of APPL1-positive vesicles. Example images of endogenous APPL1 and fluorescent Tf at 3.5-min chase after the 30-s internalization pulse in control and clathrin-depleted cells (D). Inset presents full image, yellow rectangle depicts zoomed part. The numbers of vesicles marked by APPL1 (red), Tf (blue) or EGF (green) (E) and their integral intensities (F) are plotted (quantifications based on 80 images and ∼320,000 APPL1 endosomes). (G–I) Dynasore treatment (from 10 to 60 min) does not affect the number of APPL1-positive vesicles but progressively suppresses Tf uptake (10 min of Tf internalization). (G) Example images of HeLa cells treated with Dynasore (80 µM) for 60 min. The numbers of vesicles marked by APPL1 (red) and Tf (blue) (H) and their integral intensities (I) in cells pretreated with Dynasore for the indicated times are plotted (quantifications based on 10 images, ∼110 cells, and ∼45,000 APPL1 endosomes). Bars: (D and G, inset) 10 µm.