Consequences of ESCRT knockdown on endosomal perimeter size and Cav-mRFP distribution. (A) Real-time qPCR analysis of RNAi for ESCRT complexes in S2 cells. The S2 cells were treated with dsRNA for each ESCRT component and mRNA expression of the gene was analyzed by real-time qPCR to compare with the expression in parental S2 cells. (B) Images of human Caveolin-1-mRFP distribution on GFP-2xFYVE-positive endosomes. Cav-mRFP localizes to the intraluminal space in control cells but knocking down ESCRT components by RNAi changes Cav-mRFP distribution to the limiting membrane, and enlarges endosomes. (C) Mean perimeter size of GFP-2xFYVE-positive endosomes in ESCRT KD cells. (D) The ratio of Cav-mRFP intensity on limiting membrane/in intraluminal space of endosome. In A, C, and D, *, **, and *** indicate P < 0.05, 0.01, and 0.001, respectively, compared with control cells, by two-tailed t-test. Error bars represent SEM. Sample numbers are indicated in the figure.