Fig 2: 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

Fig 3: sEV preparations from glutamine-depleted HCT116 colorectal cancer cells contain elevated levels of accessory ESCRT-III proteins. Five paired protein samples made from sEVs isolated by differential ultracentrifugation from glutamine-depleted and glutamine-replete HCT116 cells were subjected to TMT-labelled comparative proteomics analysis. (a) Volcano plot showing mean log2 fold change in specific protein levels in sEV preparations from glutamine-depleted conditions versus glutamine-replete conditions plotted against -log10 p value for null hypothesis that levels are unchanged. Coloured data points represent proteins that are significantly altered in levels between the two conditions, with colours representing number of peptides detected. (b) Heat map showing change in levels of core and accessory ESCRT proteins in five paired sEV samples (labelled 1 to 5) from glutamine-depleted versus glutamine-replete HCT116 cells. Red shows an increase in protein levels and blue indicates a reduction following glutamine depletion. (c) Western blot of sEV preparations confirms that accessory ESCRT-III proteins CHMP1A, CHMP1B and IST1 are all increased in sEVs produced by glutamine-depleted (0.15 mM) HCT116 cells. Bar chart shows protein levels and sEV number normalised to cell lysate. Data derived from five independent experiments and analysed by the Kruskal-Wallis test. Significantly changed levels are denoted by a blue (decreased) and red (increased) asterisks. *p < 0.05; n.s. = not significant. (d) Nanosight Tracking Analysis of sEV size and number for samples produced as in (c)

Fig 4: Both core and accessory ESCRT-III components regulate exosome biogenesis in non-acidic compartments of SCs. Panels A-F show basal wide-field fluorescence views of living SCs expressing Btl-GFP (green; control schematic, Figure 2a, third panel). 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). Transverse image projections of AG lumens are shown in A’’-F’’. (a) Control SC expressing rosy-RNAi. Btl-GFP-positive vesicle membranes are observed inside nearly 70% of large non-acidic compartments (arrowheads; A’) and as puncta in AG lumen (A’’). (b) SC expressing Chmp2-RNAi. Non-acidic compartment number is increased (g). Btl-GFP-positive ILVs (B’ bottom, H) and puncta in the AG lumen are reduced (B’’, I), as are DCGs. Note the large lysosomes in surrounding main cells (B Projection), which are sometimes observed in glands containing ESCRT-knockdown SCs. (c) SC expressing Chmp1-RNAi. In this cell, non-acidic compartment number is increased (g). Btl-GFP-positive ILVs, DCGs and puncta in the AG lumen are reduced (C’’, I). (d) SC expressing Chmp5-RNAi. Btl-GFP-positive ILVs and puncta in the AG lumen are reduced (D’’, I). (e) SC expressing Ist1-RNAi. Btl-GFP-positive ILVs, DCGs and puncta in the AG lumen are reduced (E’’, I). (f) SC expressing Vps4-RNAi. Btl-GFP-positive compartments are greatly increased. Btl-GFP-positive ILVs, DCGs and puncta in the AG lumen are reduced (F’’, I). (g) Bar chart showing the number of non-acidic Btl-GFP-positive compartments with diameter > 0.4 µm per SC in control vs ESCRT-III knockdowns. Note that accessory ESCRT-III knockdowns do not significantly affect overall compartment organisation and morphology, unlike Chmp2 and Vps4 knockdown. n = 30 SCs. (h) Bar chart showing percentage of Btl-GFP-positive compartments containing Btl-GFP-positive ILVs. n = 30 SCs. (i) Bar chart showing Btl-GFP fluorescent puncta number in the lumen of AGs with ESCRT-III knockdowns compared to controls. n = 10 AG lumens.All data are from 6-day-old male flies shifted to 29°C at eclosion to induce expression of transgenes. Genotypes are: w; P[w+, tub-GAL80ts]/+; dsx-GAL4/P[w+, UAS-btl-GFP]/+ with UAS-rosy-RNAi knockdown construct (a), UAS-Chmp2-RNAi-#1 (b), UAS-Chmp1-RNAi-#1 (c), UAS-Chmp5-RNAi-#1 (d), UAS-Ist1-RNAi-#1 (e), UAS-Vps4-RNAi-#1 (f). Scale bars in A-F and A’’-F’’, 10 µm, and in A’-F’, 1 µm. Data were analysed by Kruskal-Wallis test. *p < 0.05, ***p < 0.001 and ****p < 0.0001 relative to control. See Figure S4a-c for additional RNAi lines tested