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. 2023 Mar 5;12(3):12311. doi: 10.1002/jev2.12311

FIGURE 4.

FIGURE 4

ESCRT‐III knockdown inhibits exosome biogenesis in non‐acidic SC compartments, but does not change compartment identity. Panels a‐f show basal wide‐field fluorescence views of living SCs expressing YFP‐Rab11 from its endogenous genomic location (yellow). SC outline approximated by dashed white circles. Acidic compartments are marked by LysoTracker® Red (magenta). Boxed non‐acidic compartments are magnified in A’‐F’ Zoom. (a) Control SC expressing rosy‐RNAi. Rab11‐positive ILV puncta are present inside approximately 50% of Rab11‐compartments (arrowheads; Zoom). (b) In SCs expressing Chmp2‐RNAi, YFP‐Rab11‐compartment number is increased (g), but YFP‐Rab11‐positive ILVs are strongly reduced (h) and DCGs modestly reduced. (c) In SCs expressing Chmp1‐RNAi, YFP‐Rab11‐compartment number is increased (g). YFP‐Rab11‐positive ILVs are reduced (h), but DCGs are not affected. (d) In SCs expressing Chmp5‐RNAi, YFP‐Rab11‐compartment number is unchanged (g). YFP‐Rab11‐positive ILVs are reduced (h), but DCGs are not affected. (e) In SCs expressing Ist1‐RNAi, YFP‐Rab11‐compartment number is unchanged (g). YFP‐Rab11‐positive ILVs are reduced (h), but DCGs are not affected. (f) In SCs expressing Vps4‐RNAi, YFP‐Rab11‐compartment number is greatly increased (g). YFP‐Rab11‐positive ILVs and DCGs are greatly reduced (h). (g) Bar chart showing the number of non‐acidic YFP‐Rab11‐positive compartments with diameter > 0.4 μm per SC in control vs ESCRT knockdowns. n = 30. (h) Bar chart showing percentage of YFP‐Rab11 compartments containing YFP‐Rab11‐positive ILV puncta. n = 30.All data are from 6‐day‐old males shifted to 29°C at eclosion to induce transgene expression. Genotypes are: w; P[w+, tub‐GAL80ts]/+; dsx‐GAL4; TI{TI}Rab11EYFP/+ with RNAi #1 lines for each gene. Scale bars in A‐F, 10 μm and in A’‐F’, 1 μm. Data were analysed by Kruskal‐Wallis test. ***p < 0.001 and ****p < 0.0001 relative to control. See also Figure S5 for images of gene knockdowns not depicted here, and Figure S4d, S4e for additional RNAi lines tested