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Fig. 4. 2-AG is specifically sorted into microvesicles in a DAGL- and Arf6-dependent process. (A and B) Fold-enrichment (ATP/Vehicle) of lipids in EVs (A) or cells (B) following vehicle (MQ) or ATP-treatment for 30 min at 37 °C. (C) Size distribution of EVs determined by nanoparticle tracking analysis (NTA) (n = 3 videos). (D) Representative western blot and (E) quantification of CD9 signal intensity in EVs (n = 9). (F and G) 2-AG levels in EVs (F) and cells (G) relative to vehicle-treated control (n = 10/7 DMSO/DH376). (H) Size distribution of EVs determined by nanoparticle tracking analysis (NTA) (n = 3 videos). (I) Representative western blot and (J) quantification of CD9 signal intensity in EVs (n = 7). (K and L) 2-AG levels in EVs (K) and cells (L) relative to vehicle-treated control (n = 3). Cells were treated with 1 µM DH376 (C–G) or 10 µM <t>SecinH3</t> (H–L) for 20 min prior to vehicle (MQ) or ATP-treatment for 30 min at 37 °C, followed by EV isolation. Data are shown as mean ± SD. n is individual biological replicates/EV isolations. Statistical testing was performed using one-way ANOVA with Tukey’s correction for multiple comparisons. *P < 0.05, **P < 0.01, ***P < 0.001, ns not significant P > 0.05.
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Fig. 4. 2-AG is specifically sorted into microvesicles in a DAGL- and Arf6-dependent process. (A and B) Fold-enrichment (ATP/Vehicle) of lipids in EVs (A) or cells (B) following vehicle (MQ) or ATP-treatment for 30 min at 37 °C. (C) Size distribution of EVs determined by nanoparticle tracking analysis (NTA) (n = 3 videos). (D) Representative western blot and (E) quantification of CD9 signal intensity in EVs (n = 9). (F and G) 2-AG levels in EVs (F) and cells (G) relative to vehicle-treated control (n = 10/7 DMSO/DH376). (H) Size distribution of EVs determined by nanoparticle tracking analysis (NTA) (n = 3 videos). (I) Representative western blot and (J) quantification of CD9 signal intensity in EVs (n = 7). (K and L) 2-AG levels in EVs (K) and cells (L) relative to vehicle-treated control (n = 3). Cells were treated with 1 µM DH376 (C–G) or 10 µM <t>SecinH3</t> (H–L) for 20 min prior to vehicle (MQ) or ATP-treatment for 30 min at 37 °C, followed by EV isolation. Data are shown as mean ± SD. n is individual biological replicates/EV isolations. Statistical testing was performed using one-way ANOVA with Tukey’s correction for multiple comparisons. *P < 0.05, **P < 0.01, ***P < 0.001, ns not significant P > 0.05.
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Fig. 4. 2-AG is specifically sorted into microvesicles in a DAGL- and Arf6-dependent process. (A and B) Fold-enrichment (ATP/Vehicle) of lipids in EVs (A) or cells (B) following vehicle (MQ) or ATP-treatment for 30 min at 37 °C. (C) Size distribution of EVs determined by nanoparticle tracking analysis (NTA) (n = 3 videos). (D) Representative western blot and (E) quantification of CD9 signal intensity in EVs (n = 9). (F and G) 2-AG levels in EVs (F) and cells (G) relative to vehicle-treated control (n = 10/7 DMSO/DH376). (H) Size distribution of EVs determined by nanoparticle tracking analysis (NTA) (n = 3 videos). (I) Representative western blot and (J) quantification of CD9 signal intensity in EVs (n = 7). (K and L) 2-AG levels in EVs (K) and cells (L) relative to vehicle-treated control (n = 3). Cells were treated with 1 µM DH376 (C–G) or 10 µM <t>SecinH3</t> (H–L) for 20 min prior to vehicle (MQ) or ATP-treatment for 30 min at 37 °C, followed by EV isolation. Data are shown as mean ± SD. n is individual biological replicates/EV isolations. Statistical testing was performed using one-way ANOVA with Tukey’s correction for multiple comparisons. *P < 0.05, **P < 0.01, ***P < 0.001, ns not significant P > 0.05.
Secinh3 S7685, supplied by Selleck Chemicals, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Fig. 4. 2-AG is specifically sorted into microvesicles in a DAGL- and Arf6-dependent process. (A and B) Fold-enrichment (ATP/Vehicle) of lipids in EVs (A) or cells (B) following vehicle (MQ) or ATP-treatment for 30 min at 37 °C. (C) Size distribution of EVs determined by nanoparticle tracking analysis (NTA) (n = 3 videos). (D) Representative western blot and (E) quantification of CD9 signal intensity in EVs (n = 9). (F and G) 2-AG levels in EVs (F) and cells (G) relative to vehicle-treated control (n = 10/7 DMSO/DH376). (H) Size distribution of EVs determined by nanoparticle tracking analysis (NTA) (n = 3 videos). (I) Representative western blot and (J) quantification of CD9 signal intensity in EVs (n = 7). (K and L) 2-AG levels in EVs (K) and cells (L) relative to vehicle-treated control (n = 3). Cells were treated with 1 µM DH376 (C–G) or 10 µM SecinH3 (H–L) for 20 min prior to vehicle (MQ) or ATP-treatment for 30 min at 37 °C, followed by EV isolation. Data are shown as mean ± SD. n is individual biological replicates/EV isolations. Statistical testing was performed using one-way ANOVA with Tukey’s correction for multiple comparisons. *P < 0.05, **P < 0.01, ***P < 0.001, ns not significant P > 0.05.

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: The endocannabinoid 2-arachidonoylglycerol is released and transported on demand via extracellular microvesicles.

doi: 10.1073/pnas.2421717122

Figure Lengend Snippet: Fig. 4. 2-AG is specifically sorted into microvesicles in a DAGL- and Arf6-dependent process. (A and B) Fold-enrichment (ATP/Vehicle) of lipids in EVs (A) or cells (B) following vehicle (MQ) or ATP-treatment for 30 min at 37 °C. (C) Size distribution of EVs determined by nanoparticle tracking analysis (NTA) (n = 3 videos). (D) Representative western blot and (E) quantification of CD9 signal intensity in EVs (n = 9). (F and G) 2-AG levels in EVs (F) and cells (G) relative to vehicle-treated control (n = 10/7 DMSO/DH376). (H) Size distribution of EVs determined by nanoparticle tracking analysis (NTA) (n = 3 videos). (I) Representative western blot and (J) quantification of CD9 signal intensity in EVs (n = 7). (K and L) 2-AG levels in EVs (K) and cells (L) relative to vehicle-treated control (n = 3). Cells were treated with 1 µM DH376 (C–G) or 10 µM SecinH3 (H–L) for 20 min prior to vehicle (MQ) or ATP-treatment for 30 min at 37 °C, followed by EV isolation. Data are shown as mean ± SD. n is individual biological replicates/EV isolations. Statistical testing was performed using one-way ANOVA with Tukey’s correction for multiple comparisons. *P < 0.05, **P < 0.01, ***P < 0.001, ns not significant P > 0.05.

Article Snippet: SecinH3 (S7685), ML- 7 (S8388), GW4896 (S7609), and Sotrastaurin (S2791) were purchased from Selleck Chemicals.

Techniques: Western Blot, Control, Isolation