Deletion strains tested for their ability to relocate mCherry-D4H upon CK-666 treatment. (A) Listed deletion strains expressing mCherry-D4H were treated with CK-666 for 1 h and evaluated by spinning disc microscopy. All strains were capable of internalizing mCherry-D4H. (B) The deletion of arf6 and vps1, encoding proteins predicted to be involved in nonclathrin mediated endocytosis, does not block mCherry-D4H internalization. arf6Δ and vps1Δ mutants expressing mCherry-D4H were incubated in the presence of 500 µM CK-666 for 1 h and evaluated by spinning disk microscopy. Both strains translocated the sterol biosensor D4H upon the treatment. Scale bar, 2 µm.