Figure S1. Confirmation and additional... | Rockefeller University Press

Figure S1.

Confirmation and additional characterization of Rop, Vps20, and VhaPPA1-1 hits. (A) Confocal images of fat body from wild-type, BM-40-SPARC>Tango1i, >Rab1i, Ropi (THU0467), >Vps20i (v47653, and >VhaPPA1-1i (v47188) L3 larvae showing intracellular accumulation of Collagen IV (α2 chain, Vkg-GFP). Knockdown of Rop, Vps20, or VhaPPA1-1 results in diffuse intracellular accumulation using RNAi transgenic constructs alternative to the ones in the initial screening (Ke et al., 2018). (B) Quantification of Collagen IV accumulation in fat body of the indicated genotypes, measured in images like those in A. Each dot represents total Collagen IV–GFP intensity measured in one cell (n = 10 cells per genotype). Horizontal lines mark mean values. Differences with the wild type were significant in unpaired t tests (P < 0.0001). (C) Confocal images of fat body from BM-40-SPARC-GAL4+GAL80ts, BM-40-SPARC+GAL80ts>Ropi (v19696), >Vps20i (THU5269), and >VhaPPA1-1i (v47188) L3 larvae (feeding stage) showing localization of Collagen IV (α2 chain Vkg-GFP, green) and BM-40-SPARC-GAL4–driven autophagy marker Atg8a-mCherry (magenta). Nuclei in cyan (DAPI). Larvae were dissected 2 d after knockdown initiation (see Materials and methods). Magnified insets in lower right corners show accumulation of Collagen IV distinct from Atg8a puncta in mutant conditions. (D) Confocal images of fat body from wild-type L3 larvae (wandering stage), and BM-40-SPARC>Ropi (v19696), >Vps20i (THU5269), and >VhaPPA1-1i (v47188) L3 larvae (feeding stage) showing lysosomal marker Lamp1-YFP (green) and acidification stain LysoTracker Red (magenta, separate channel below). Acidification of autolysosomes is reduced in mutant conditions (compare also TEM images in Figs. 1 E and 9 F). (E) LysoTracker intensity measured in images like those in D. The data are presented as violin plots where quartiles are marked, with dots representing individual measurements of vesicle LysoTracker intensity. n = 121, 265, 265, and 100, respectively. (F) Plots of LysoTracker intensity vs. vesicle size, measured in images like those in D. Dots represent individual measurements of vesicle LysoTracker intensity (same data as in E) and area in section. Graphs additionally depict linear regression and 99% confidence intervals. The same wild-type data is plotted in all three graphs.

Confirmation and additional characterization of Rop, Vps20, and VhaPPA1-1 hits. (A) Confocal images of fat body from wild-type, BM-40-SPARC>Tango1ⁱ, >Rab1ⁱ, Ropⁱ (THU0467), >Vps20ⁱ (v47653, and >VhaPPA1-1ⁱ (v47188) L3 larvae showing intracellular accumulation of Collagen IV (α2 chain, Vkg-GFP). Knockdown of Rop, Vps20, or VhaPPA1-1 results in diffuse intracellular accumulation using RNAi transgenic constructs alternative to the ones in the initial screening (Ke et al., 2018). (B) Quantification of Collagen IV accumulation in fat body of the indicated genotypes, measured in images like those in A. Each dot represents total Collagen IV–GFP intensity measured in one cell (n = 10 cells per genotype). Horizontal lines mark mean values. Differences with the wild type were significant in unpaired t tests (P < 0.0001). (C) Confocal images of fat body from BM-40-SPARC-GAL4+GAL80^ts, BM-40-SPARC+GAL80^ts>Ropⁱ (v19696), >Vps20ⁱ (THU5269), and >VhaPPA1-1ⁱ (v47188) L3 larvae (feeding stage) showing localization of Collagen IV (α2 chain Vkg-GFP, green) and BM-40-SPARC-GAL4–driven autophagy marker Atg8a-mCherry (magenta). Nuclei in cyan (DAPI). Larvae were dissected 2 d after knockdown initiation (see Materials and methods). Magnified insets in lower right corners show accumulation of Collagen IV distinct from Atg8a puncta in mutant conditions. (D) Confocal images of fat body from wild-type L3 larvae (wandering stage), and BM-40-SPARC>Ropⁱ (v19696), >Vps20ⁱ (THU5269), and >VhaPPA1-1ⁱ (v47188) L3 larvae (feeding stage) showing lysosomal marker Lamp1-YFP (green) and acidification stain LysoTracker Red (magenta, separate channel below). Acidification of autolysosomes is reduced in mutant conditions (compare also TEM images in Figs. 1 E and 9 F). (E) LysoTracker intensity measured in images like those in D. The data are presented as violin plots where quartiles are marked, with dots representing individual measurements of vesicle LysoTracker intensity. n = 121, 265, 265, and 100, respectively. (F) Plots of LysoTracker intensity vs. vesicle size, measured in images like those in D. Dots represent individual measurements of vesicle LysoTracker intensity (same data as in E) and area in section. Graphs additionally depict linear regression and 99% confidence intervals. The same wild-type data is plotted in all three graphs.