Sterols are internalized to endosomes. (A and B) Correlative TEM and epifluorescence (mCherry-D4H) images of a 300-nm section of mCherry-D4H–expressing cells treated with CK-666 for 45 min to 1 h. The overlay is shown on the left. In A, the TEM image is a composite of three micrographs (junctions indicated by dashed lines). Virtual sections through tomographic reconstruction of the four regions highlighted by gray boxes are shown on the right. In B, serial virtual sections at 24.1 nm distance through tomographic reconstruction of the boxed region are shown on the right. Yellow arrowheads point to D4H-positive spherical organelles. (C and D) Colocalization of mCherry-D4H and Sec72-GFP (C) or GFP-Syb1 (D) in cells with or without CK-666 for 1 h. An example time-lapse sequence is shown on the right. 13% of D4H internal dots overlapped with Sec72-GFP and 83.5% with GFP-Syb1 upon CK-666 treatment (n = 20 cells). (E) Colocalization of internal sfGFP-D4H (sfGFP-superfolder GFP) structures with internalized FM4-64 in cells prelabeled with FM4-64 and treated 30 min with CK-666 (left) or labeled with FM4-64 upon CK-666 wash-out (right). (F) Untreated WT interphase cells with internal mCherry-D4H signal, which colocalizes with GFP-Syb1. Scale bars, 200 nm in A and B; 1 µm in D, right panel; 0.5 µm in C, right panel; and 2 µm elsewhere.