selective pld1 inhibitor cay10593  (Cayman Chemical)

Journal: eLife

Article Title: Ral GTPases promote breast cancer metastasis by controlling biogenesis and organ targeting of exosomes

doi: 10.7554/eLife.61539

Figure Lengend Snippet: ( a ) PLD1 and PLD2 expression in 4T1 shControl cells quantified by qRT-PCR and normalized by gapdh expression. ( b ) Representative confocal images showing PLD1-GFP and PLD2-GFP subcellular localization in 4T1 cells stained with lysotracker. Scale bars: 10 μm ( c ) Representative confocal images of PLD2-GFP localization in shControl, shRalA, and shRalB cells. Scale bar: 10 μm ( d ) PA/PC ratio of species known to be targeted by PLD1 identified in extracellular vesicles (EVs) isolated from shControl, shRalA, and shRalB cells. Each dot represents one experiment; three independent experiments.

Article Snippet: Cells were incubated with the following drugs in the appropriate medium: RalA/B inhibitors BQU57 (10 μM; Sigma) and RBC8 (10 μM; Sigma), PLD1 inhibitor CAY10593 (10 μM; Santa Cruz Biotechnology) or PLD2 inhibitor CAY10594 (10 μM; Santa Cruz Biotechnology).

Techniques: Expressing, Quantitative RT-PCR, Staining, Isolation

Journal: eLife

Article Title: Ral GTPases promote breast cancer metastasis by controlling biogenesis and organ targeting of exosomes

doi: 10.7554/eLife.61539

Figure Lengend Snippet: ( a ) Representative confocal images of 4T1 cells co-transfected with PLD1-GFP and tdTomato-RalA (upper panels) or tdTomato-RalB (lower panels) and incubated with Lysotracker. Scale bar: 10 μm; zoom: 2 μm. ( b ) Electron microscopy analysis of 4T1 cells treated with PLD1 or PLD2 inhibitor. Scale bar: 1 μm. Violin plots show quantification of the number of multi-vesicular body (MVB) per cytoplasmic surface. Each dot represents one field of view; horizontal bar represents the average (180–194 fields of view; Kruskal-Wallis test followed by Dunn's Multiple Comparison Test). ( c ) Nanoparticle tracking analysis of extracellular vesicles (EVs) isolated by ultracentrifugation (100,000 g pellet) from the supernatant of 4T1 cells treated with PLD1 (CAY10593) or PLD2 (CAY10594) inhibitor. Each dot represents one experiment (three independent experiments; One-Way Anova permutation test followed by fdr multi-comparison permutation test). ( d ) Representative confocal images of shControl, shRalA and shRalB 4T1 cells transfected with PLD1-GFP. Scale bar: 10 μm; zoom: 2 μm. Graph shows the percentage of cells with high (>5) number of PLD1-GFP cytoplasmic puncta. (Each dot represents one experiment. Five independent experiments; Number of cells analyzed: shCtl (136), shRalA (170), shRalB (244); Kruskal-Wallis test followed by Dunn's Multiple Comparison Test). ( e ) Quantification of the Phosphatidic Acid (PA) / PhosphatidylCholine (PC) ratio in EVs isolated from shControl, shRalA, and shRalB cells (each dot represents one experiment; three independent experiments; One-Way Anova permutation test followed by fdr multi-comparison permutation test; fdr <0.1). ( f ) Model showing how RalA and RalB could control PLD1 localization on MVBs, thereby inducing the PA accumulation on MVBs, promoting MVB homeostasis and controlling exosome secretion.

Article Snippet: Cells were incubated with the following drugs in the appropriate medium: RalA/B inhibitors BQU57 (10 μM; Sigma) and RBC8 (10 μM; Sigma), PLD1 inhibitor CAY10593 (10 μM; Santa Cruz Biotechnology) or PLD2 inhibitor CAY10594 (10 μM; Santa Cruz Biotechnology).

Techniques: Transfection, Incubation, Electron Microscopy, Comparison, Isolation, Control