Figure 2. LLOMe induces LC3 recruitment... | Rockefeller University Press

Figure 2.

$LLOMe induces LC3 recruitment directly to lysosomes and their tubulation. (A) Representative confocal images of MCF10A cells expressing GFP-LC3A, treated with LLOMe (250 μM, 20 min) or PP242 (1 μM, 1 h) and stained for LAMP1. Colocalization analysis is shown. Scale bar, 5 μm. (B) Quantification of GFP-LC3A overlap with LAMP1. Eight fields of view from two experiments were analyzed; ****, P < 0.0001, **, P < 0.004, unpaired t test. (C) Western blot analysis of input and LysoIP fractions from control or ATG13 KO cells expressing TMEM192-3xHA treated with LLOMe (250 μM, 20 min). (D) Images from time-lapse confocal microscopy before and after 20 min LLOMe (250 μM) treatment. Inserts show two areas of LLOMe treated cells. Arrows denote GFP-LC3A positive tubules. Scale bar, 30 μm. (E) Images from time-lapse series showing the formation of GFP-LC3A puncta following LLOMe (250 μM) treatment (red arrow) and subsequent tubulation. min:s. Scale bar, 3 μm. (F) MCF10A cells coexpressing GFP-LC3A and LAMP1-RFP treated with LLOMe (250 μM). Images show three examples (i, ii, iii). Arrows denote puncta and arrowheads denote tubules. Scale bar, 2 μm. (G) Time-lapse confocal images of LLOMe-induced GFP-LC3A tubulation. Three examples are shown (i, ii, iii), red arrows denote the vesiculation of tubules. Scale bar, 2 μm. (H) Quantification of tubules in cells treated with LLOMe (250 μM). Number of tubules per cell were analyzed in five fields of view from cells pretreated with nocodazole (1 μM), Vps34 IN-1 (5 μM), or LRRK2 inhibitor MLi2 (1 μM). A total of >150 cells per condition were analyzed. Source data are available for this figure: SourceData F2.$

LLOMe induces LC3 recruitment directly to lysosomes and their tubulation. (A) Representative confocal images of MCF10A cells expressing GFP-LC3A, treated with LLOMe (250 μM, 20 min) or PP242 (1 μM, 1 h) and stained for LAMP1. Colocalization analysis is shown. Scale bar, 5 μm. (B) Quantification of GFP-LC3A overlap with LAMP1. Eight fields of view from two experiments were analyzed; ****, P < 0.0001, **, P < 0.004, unpaired t test. (C) Western blot analysis of input and LysoIP fractions from control or ATG13 KO cells expressing TMEM192-3xHA treated with LLOMe (250 μM, 20 min). (D) Images from time-lapse confocal microscopy before and after 20 min LLOMe (250 μM) treatment. Inserts show two areas of LLOMe treated cells. Arrows denote GFP-LC3A positive tubules. Scale bar, 30 μm. (E) Images from time-lapse series showing the formation of GFP-LC3A puncta following LLOMe (250 μM) treatment (red arrow) and subsequent tubulation. min:s. Scale bar, 3 μm. (F) MCF10A cells coexpressing GFP-LC3A and LAMP1-RFP treated with LLOMe (250 μM). Images show three examples (i, ii, iii). Arrows denote puncta and arrowheads denote tubules. Scale bar, 2 μm. (G) Time-lapse confocal images of LLOMe-induced GFP-LC3A tubulation. Three examples are shown (i, ii, iii), red arrows denote the vesiculation of tubules. Scale bar, 2 μm. (H) Quantification of tubules in cells treated with LLOMe (250 μM). Number of tubules per cell were analyzed in five fields of view from cells pretreated with nocodazole (1 μM), Vps34 IN-1 (5 μM), or LRRK2 inhibitor MLi2 (1 μM). A total of >150 cells per condition were analyzed. Source data are available for this figure: SourceData F2.