Figure 2. Retromer affects lysosomal sensitivity to damage.

(A, B) High content microscopy (HCM) imaging and quantification of Gal3 response (puncta/cell of endogenous Gal3 profiles stained for immunofluorescence) in HeLa^WT, HeLa^ATG5-KO, and HeLa^VPS35-KO cells subjected to lysosomal damage by Leu-Leu-O-Me ester hydrobromide (LLOMe; 2 mM, 30 min). HCM, an unbiased machine-driven image acquisition and data analysis based on presets of >500 valid primary objects/cells per well (representative images shown; white mask, cell; red masks, Gal3 puncta), with a minimum of 5 wells per sample (sampling error), and n ≥ 3, independent biological replicates (experimental error) in separate 96-well plates. Data, means ± SE (n = 3), two-way ANOVA with Tukey’s multiple comparisons. (C, D) Complementation analysis of VPS35^KO LyHYP (lysosome hypersensitivity) phenotype monitored by HCM quantification of Gal3 puncta in HeLa^VPS35-KO cells transfected with GFP (control) or GFP-VPS35 expressing plasmids. Data, means ± SE (n = 3); one-way ANOVA with Tukey’s multiple comparisons. (E, F) Comparative HCM analysis of ubiquitin (immunofluorescence; FK2 antibody) response to lysosomal damage (LLOMe; 2 mM, 30 min) in HeLa^ATG5-KO and HeLa^VPS35-KO cells. Yellow profiles, colocalization of ubiquitin and LAMP1. Top panels, ubiquitin immunostaining alone. Data, means ± SE (n = 4), two-way ANOVA with Tukey’s multiple comparisons. (G, H) HCM quantification of ALIX localization to endolysosomal compartments (% of LAMP1 profiles positive for ALIX immunostaining) in Huh7^WT, Huh7^ATG5-KO, and Huh7^VPS35-KO cells following lysosomal damage. Yellow profiles, colocalization of ALIX and LAMP1. Data, means ± SE (n = 3); two-way ANOVA with Tukey’s multiple comparisons. HCM images in all relevant panels, examples from a bank of unbiased operator-independent machine-collected and algorithm-processed fields containing a minimum of 500 primary objects/cells per well (5 wells minimum per 96-well plate; 3 plates minimum), per cell line/condition.

Figure 2—figure supplement 1. Retromer affects a subset of responses to lysosomal damage.

(A, B) High content microscopy (HCM) imaging and quantification of LC3 (puncta/cell of immunofluorescently stained endogenous LC3 profiles) in HeLa^WT, and HeLa^VPS35-KO cells in response to lysosomal damage by LLOMe. Data, means ± SE (n = 3), one-way ANOVA with Tukey’s multiple comparisons. (C, D) HCM analysis of ubiquitin (immunofluorescence; FK2 antibody) response to lysosomal damage (LLOMe; 1 mM, 2 hr) in Huh7^WT, Huh7^ATG5-KO, and Huh7^VPS35-KO cells. Yellow profiles, colocalization of ubiquitin and LAMP1. Data, means ± SE (n = 3), one-way ANOVA with Tukey’s multiple comparisons. (E, F) HCM quantification of ALIX localization to endolysosomal compartments (% of LAMP1 profiles positive for ALIX immunostaining) in HeLa^WT and HeLa^VPS35-KO cells following lysosomal damage. Yellow profiles, colocalization of ALIX and LAMP1. Data, means ± SE (n = 3); two-way ANOVA with Tukey’s multiple comparisons. HCM images in all relevant panels, examples from a bank of unbiased operator-independent machine-collected and algorithm-processed fields containing a minimum of 500 primary objects/cells per well (5 wells minimum per 96-well plate; 3 plates minimum), per cell line/condition.