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MedChemExpress
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Tocris
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Tocris
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ChemScene llc
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ChemScene llc
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Journal: Journal of Extracellular Vesicles
Article Title: Cholesterol Deficiency Directs Autophagy‐Dependent Secretion of Extracellular Vesicles
doi: 10.1002/jev2.70218
Figure Lengend Snippet: Increased sEV secretion in cholesterol‐depleted conditions is due to autophagy‐driven signalling. (A) Representative images for CD63 and LC3B expression in HEK293T or HEK293T ATG2A/B KO cells after vehicle, AY9944 or rapamycin and chloroquine (Rap & CQN) treatment. Scale bar, 25 µm. (B) Quantified CD63 expression in HEK293T and HEK293T ATG2A/B KO cells (mean ± SEM; n = 8 images analyzed from 2 independent experiments). Two‐way ANOVA (treatment effect: F2,42 = 47.99, p < 0.0001; Autophagy effect: F1,42 = 5.422, p ≤ 0.0248). Sidak's multiple comparisons test (** p ≤ 0.005, **** p < 0.0001). (C) LC3B expression in HEK293T and HEK293T ATG2A/B KO cells (mean ± SEM; n = 8, 4 images analyzed from 2 independent experiments). Two‐way ANOVA (interaction effect: F2,42 = 6.126, p ≤ 0.0046; treatment effect: F2,42 = 5.995, p ≤ 0.0051; Autophagy effect: F1,42 = 12.65, p ≤ 0.0009). Sidak's multiple comparisons test (* p ≤ 0.05, ** p ≤ 0.005, *** p ≤ 0.001). (D) Immunoblot for LC3B in isolated sEVs upon cholesterol depletion. (E) CD63 and LC3B colocalization in HEK293T and HEK293T ATG2A/B KO cells (mean ± SEM; n = 8 images analyzed from 2 independent experiments). Two‐way ANOVA (interaction effect: F2,53 = 128.9, p < 0.0001; treatment effect: F2,53 = 157.0, p < 0.0001; autophagy effect: F1,53 = 453.1, p < 0.0001). Sidak's multiple comparisons test (**** p < 0.0001). (F) Quantified sEV secretion in HEK293T or HEK293T ATG2A/B KO cells following vehicle, AY9944, or Rap & CQN treatments (mean ± SEM; n = 3 biological replicates from 3 independent experiments). One‐way ANOVA (F2,6 = 56.17, p ≤ 0.0001), Dunnett's multiple comparisons test (** p ≤ 0.01, **** p < 0.0001). (G) sEV secretion in HEK293T or HEK293T ATG2A/B KO cells following vehicle or D609 treatments (mean ± SEM; n = 3 biological replicates from 3 independent experiments). Unpaired t‐test (HEK293T: t4 = 9.395, *** p ≤ 0.0007; HEK293T ATG2A/B KO: t 4 = 4.069, ** p ≤ 0.0152).
Article Snippet: For FBS rescue experiments, HEK293T cells were treated with vehicle, simvastatin or AY9944 for 24 h. Media was then replaced with 10% EV‐depleted FBS for 24 or 48 h. Other pharmacological agents utilized in these studies include rapamycin (1 μM, Selleck Chemicals; mTOR inhibitor), chloroquine (10 μM, Cayman Chemical; inhibits autophagosome‐lysosome fusion),
Techniques: Expressing, Western Blot, Isolation
Journal: The Journal of Biological Chemistry
Article Title: Multiple activities of sphingomyelin synthase 2 generate saturated fatty acid– and/or monounsaturated fatty acid–containing diacylglycerol
doi: 10.1016/j.jbc.2024.107960
Figure Lengend Snippet: Effects of D609, a PC-PLC/SMS inhibitor, on PC-PLC, PE-PLC, SMS, and CPES activities of SMS2. A , DG-generating activities of SMS2, which was purified in the presence of detergents, were analyzed in the presence of various concentrations of D609 (0, 0.25, 0.5, and 1 mM). Purified WT SMS2-TS (approximately 500 ng) was incubated in the presence of 16:0/18:1-PC alone, 16:0/18:1-PC and d18:1/18:0-Cer, 16:0/18:1-PE alone, or 16:0/18:1-PE and d18:1/18:0-Cer–containing detergent mixed micelles for 2 h at 37 °C. Produced 16:0/18:1-DG was quantified using LC–MS/MS. Values are presented as percentages of the activity of SMS2 in the absence of D609 (set at 100%). Values are presented as the mean ± SD (n = 6, technical replicates). ∗∗∗, p < 0.005 ( versus control (without D609)). One-way ANOVA with Dunnett’s post hoc test was used. B , SM-producing activities of SMS2, which was purified in the presence of detergents, in the presence of 16:0/18:1-PC and d18:1/18:0-Cer–containing detergent mixed micelles were analyzed in the presence of various concentrations of D609 (0, 0.25, 0.5, and 1 mM). Values are presented as percentages of the SMS activity of SMS2 in the absence of D609 (set to 100%). The values are presented as the mean ± SD (n = 4, technical replicates). ∗, p < 0.05; ∗∗, p < 0.01 ( versus control (without D609)). One-way ANOVA with Dunnett’s post hoc test was used. C , CPE-producing activities of SMS2, which was purified in the presence of detergents, in the presence of 16:0/18:1-PE and d18:1/18:0-Cer–containing detergent mixed micelles were analyzed in the presence of various concentrations of D609 (0, 0.25, 0.5, and 1 mM). Values are presented as percentages of the CPES activity of SMS2 in the absence of D609 (set at 100%). Values are presented as the mean ± SD (n = 6, technical replicates). ∗∗, p < 0.01; ∗∗∗, p < 0.005 ( versus control (without D609)). One-way ANOVA with Dunnett’s post hoc test was used. Cer, ceramide; CPE, ceramide phosphoethanolamine; CPES, CPE synthase; DG, diacylglycerol; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PLC, phospholipase C; SM, sphingomyelin; SMS, sphingomyelin synthase; TS, Twin-Strep.
Article Snippet:
Techniques: Purification, Incubation, Produced, Liquid Chromatography with Mass Spectroscopy, Activity Assay, Control
Journal: The Journal of Biological Chemistry
Article Title: Multiple activities of sphingomyelin synthase 2 generate saturated fatty acid– and/or monounsaturated fatty acid–containing diacylglycerol
doi: 10.1016/j.jbc.2024.107960
Figure Lengend Snippet: Comparison of SMS2 (our findings and previous reports), SMS1, and SMSr
Article Snippet:
Techniques: Comparison, Membrane, Expressing, Activity Assay, In Vitro, Inhibition, Mutagenesis
Journal: The Journal of Biological Chemistry
Article Title: Multiple activities of sphingomyelin synthase 2 generate saturated fatty acid– and/or monounsaturated fatty acid–containing diacylglycerol
doi: 10.1016/j.jbc.2024.107960
Figure Lengend Snippet: Comparison of bacterial, plant, and mammalian PLCs
Article Snippet:
Techniques: Comparison, Activity Assay, Membrane, Inhibition