SGs induced by lysosomal damage show limited and dynamic interactions with lysosomes. (A) Quantification by HCM of G3BP1 puncta in Huh7 cells transfected with scrambled siRNA as control (SCR) or RNASET2 siRNA treated with 2 mM LLOMe for 30 min. Red masks, computer-identified G3BP1 puncta. (B) Immunofluorescence confocal microscopy analysis of G3BP1 and LAMP2. U2OS cells were treated with 2 mM LLOMe for 30 min and stained for endogenous G3BP1 and LAMP2. Scale bar, 5 µm. (C) Quantification by HCM of overlaps between G3BP1 and LAMP2 in U2OS cells treated with 2 mM LLOMe for 30 min. White masks, algorithm-defined cell boundaries. Yellow masks, computer-identified overlap of G3BP1 and LAMP2. (D) Quantification by HCM of overlaps between FLAG-NUFIP2 and LAMP2 in U2OS cells expressing FLAG-NUFIP2 treated with 2 mM LLOMe for 30 min. Yellow masks, computer-identified overlap of FLAG-NUFIP2 and LAMP2. (E) Still frames from live-cell fluorescence imaging analysis of mCherry-G3BP1 and GFP-LAMP1. U2OS cells expressing mCherry-G3BP1 and GFP-LAMP1 were incubated with 2 mM LLOMe during live-cell fluorescence imaging. Arrows, the representative regions at indicated timepoint. (F) Zoom views of regions in E. Ctrl, control (untreated cells). Data, means ± SEM; HCM: n ≥ 3 (each experiment: 500 valid primary objects/cells per well, ≥5 wells/sample). †, P ≥ 0.05 (not significant); **, P < 0.01, ANOVA. Source data are available for this figure: SourceData FS2.