The ESCRT machinery is essential for the degradation of Cot1. (A) Western blots showing the degradation of Cot1-GFP in WT and vps4Δ cells. (B) Quantification (±SD, n = 3) of protein levels in A. (C) Time-lapse imaging of Cot1-GFP in WT and vps4Δ strains during an 8-h rapamycin treatment. Both strains were grown in the same imaging chamber. Arrows highlight the ILFs in vps4Δ cells. (D) Images showing the colocalization of Cot1-GFP with Zrc1-mCherry in vps4Δ strain cells during rapamycin treatment. (E) Z stacks showing the colocalization of Cot1-GFP with Zrc1-mCherry in the inserts of D. (F) Percentage of vps4Δ cells with ILF during rapamycin treatment. Error bars represent SD. Numbers on each column indicate the total number of cells counted. The statistical analysis was performed with a paired Student t test. ****, P ≤ 0.0001. (G) Quantification of the colocalization between Cot1-GFP and Zrc1-mCherry on ILF. Scale bars, 3 µm.