Journal: bioRxiv

Article Title: Extracellular vesicles promote autophagy in human microglia through lipid raft-dependent mechanisms

doi: 10.1101/2023.07.03.547488

Figure Lengend Snippet: A – Confocal images of LC3B protein (red) in microglia cells treated with EVs for different durations (scale bar = 100 μm, magnification – 63x). B – Mean fluorescence intensity of LC3B protein per cell measured using LAS X software. Data represent the mean ± SEM from 20 fields of view (n = 4), results normalized to control. Statistical significance was determined using Kruskal-Wallis test followed by Dunn’s post-hoc test in GraphPad Prism 8.0.1 software (* p<0.05; *** p<0.001; **** p<0.0001). C – Representative confocal images of microglial cells infected with Premo™ Autophagy Tandem Sensor RFP-GFP-LC3 Kit. D – Statistical analysis of GFP-LC3B and RFP-LC3B protein expression. Data shown represent the mean ± SEM from 15 fields of view (n = 3). Statistical significance was determined using Kruskal-Wallis test followed by Dunn’s post-hoc test (** p<0.01). E – Estimation of autophagic flux by RFP:GFP ratio. Data represent the mean ± SEM from three independent experiments (n = 3). Statistical significance was determined using the Mann-Whitney U test (** p<0.01). F – Confocal images of microglial cells infected with Premo™ Autophagy Tandem Sensor RFP-GFP-LC3 Kit after exposure to EVs for different durations. G - Quantification data of GFP-LC3B and RFP-LC3B protein expression. Data shown represent the mean ± SEM from 15 fields of view (n = 3). Statistical analysis was performed using Kruskal-Wallis test followed by Dunn’s post-hoc test for GFP-LC3B ( H ) and RFP-LC3B ( I ) protein expression (* p<0.05; *** p<0.001)

Article Snippet: We used Premo™ Autophagy Tandem Sensor RFP-GFP-LC3 Kit (P36239, Molecular Probes, Thermo Fisher Scientific, Bremen, Germany) that allows visualization of the maturation of the autophagosomes to the autolysosomes.

Techniques: Fluorescence, Software, Infection, Expressing, MANN-WHITNEY

Journal: bioRxiv

Article Title: Extracellular vesicles promote autophagy in human microglia through lipid raft-dependent mechanisms

doi: 10.1101/2023.07.03.547488

Figure Lengend Snippet: A – Confocal images of labeled LC3B protein (red) in microglia cells in the presence and absence of pre-treatment with 5 mM MβCD for 1 hour (63× magnification immersion objective, scale bar = 100 μm). B – The mean fluorescence intensity of LC3B protein per cell were measured with Leica Application Suite X (LAS X) software, data shown represent the results of 15 fields of view for each experimental group from three independent biological experiments (n = 3), plotted as the mean ± SEM, results normalized to control. Statistical significance was analyzed by Kruskal–Wallis test followed by Dunn’s post-hoc test in GraphPad Prism 8.0.1 software (**** p < 0.0001)

Article Snippet: We used Premo™ Autophagy Tandem Sensor RFP-GFP-LC3 Kit (P36239, Molecular Probes, Thermo Fisher Scientific, Bremen, Germany) that allows visualization of the maturation of the autophagosomes to the autolysosomes.

Techniques: Labeling, Fluorescence, Software

Journal: bioRxiv

Article Title: Extracellular vesicles promote autophagy in human microglia through lipid raft-dependent mechanisms

doi: 10.1101/2023.07.03.547488

Figure Lengend Snippet: A – Confocal images of LC3B protein (red) in microglia cells treated with EVs (1 AU) and LPS (5 μg/ml) for 16 hours (scale bar = 100 μm, magnification – 63x). B – Mean fluorescence intensity of LC3B protein per cell measured using LAS X software. Data represents the mean ± SEM from 15 fields of view (n = 3). Statistical significance was determined using Kruskal-Wallis test followed by Dunn’s post-hoc test in GraphPad Prism 8.0.1 software (* p<0.05; **** p<0.0001). C – Confocal images of microglial cells infected with Premo™ Autophagy Tandem Sensor RFP-GFP-LC3 Kit. D – Quantification data of GFP-LC3B and RFP-LC3B protein expression. Data shown represents the mean ± SEM from 15 fields of view (n = 3). Statistical analysis was performed using Kruskal-Wallis test followed by Dunn’s post-hoc test for GFP-LC3B ( E ) and RFP-LC3B ( F ) protein expression (* p<0.05; ** p<0.01; *** p<0.001). G – Estimation of autophagic flux by RFP:GFP ratio. Data represents the mean ± SEM from three independent experiments (n = 3). Statistical significance was determined using Kruskal-Wallis test followed by Dunn’s post-hoc test (* p<0.05; *** p<0.001).

Article Snippet: We used Premo™ Autophagy Tandem Sensor RFP-GFP-LC3 Kit (P36239, Molecular Probes, Thermo Fisher Scientific, Bremen, Germany) that allows visualization of the maturation of the autophagosomes to the autolysosomes.

Techniques: Fluorescence, Software, Infection, Expressing

Journal: bioRxiv

Article Title: Extracellular vesicles promote autophagy in human microglia through lipid raft-dependent mechanisms

doi: 10.1101/2023.07.03.547488

Figure Lengend Snippet: A – confocal images of labeled LC3B protein (red) in microglia cells. The cells were treated with EVs (1 AU) and LPS (5 μg/ml) for 16 hours before immunocytochemistry and fixation (scale bar = 100 μm, magnification – 63x). B – the mean fluorescence intensity of LC3B protein per cell were measured with Leica Application Suite X (LAS X) software. Data shown represent the results of 15 fields of view for each experimental group from three independent biological experiments (n = 3), plotted as the mean ± SEM, results normalized to control. Statistical significance was analyzed by Kruskal–Wallis test followed by Dunn’s post-hoc test in GraphPad Prism 8.0.1 software (* p<0.05; ** p<0.01; **** p < 0.0001)

Article Snippet: We used Premo™ Autophagy Tandem Sensor RFP-GFP-LC3 Kit (P36239, Molecular Probes, Thermo Fisher Scientific, Bremen, Germany) that allows visualization of the maturation of the autophagosomes to the autolysosomes.

Techniques: Labeling, Immunocytochemistry, Fluorescence, Software

Journal: bioRxiv

Article Title: Extracellular vesicles promote autophagy in human microglia through lipid raft-dependent mechanisms

doi: 10.1101/2023.07.03.547488

Figure Lengend Snippet: A – Confocal images of labeled lipid rafts (red) in human microglia cells treated with a selective P2X4R antagonist 5-BDBD (scale bar = 100 μm, magnification – 63x). B – Mean fluorescence intensity of labeled ganglioside GM1 per cell measured using LAS X software. Data represents the mean ± SEM from 15 fields of view (n = 3), results normalized to control. Statistical significance was determined using Kruskal-Wallis test followed by Dunn’s post-hoc test in GraphPad Prism 8.0.1 software (*** p<0.001; **** p<0.0001). C – Confocal images of microglial cells infected with Premo™ Autophagy Tandem Sensor RFP-GFP-LC3 Kit. D – Quantification data of GFP-LC3B and RFP-LC3B protein expression. Data shown represents the mean ± SEM from 15 fields of view (n = 3). Statistical analysis was performed using Kruskal-Wallis test followed by Dunn’s post-hoc test for GFP-LC3B ( E ) and RFP-LC3B ( F ) protein expression (* p<0.05; ** p<0.01; *** p<0.001).

Article Snippet: We used Premo™ Autophagy Tandem Sensor RFP-GFP-LC3 Kit (P36239, Molecular Probes, Thermo Fisher Scientific, Bremen, Germany) that allows visualization of the maturation of the autophagosomes to the autolysosomes.

Techniques: Labeling, Fluorescence, Software, Infection, Expressing

Journal: bioRxiv

Article Title: Extracellular vesicles promote autophagy in human microglia through lipid raft-dependent mechanisms

doi: 10.1101/2023.07.03.547488

Figure Lengend Snippet: A – Confocal images of labeled LC3B protein (red) in microglia cells in the presence and absence of pre-treatment with 10 μM 5-BDBD for 30 min (63× magnification immersion objective, scale bar = 100 μm). B – The mean fluorescence intensity of LC3B protein per cell were measured with Leica Application Suite X (LAS X) software, data shown represent the results of 15 fields of view for each experimental group from three independent biological experiments (n = 3), plotted as the mean ± SEM, results normalized to control. Statistical significance was analyzed by Kruskal–Wallis test followed by Dunn’s post-hoc test in GraphPad Prism 8.0.1 software (**** p < 0.0001)

Article Snippet: We used Premo™ Autophagy Tandem Sensor RFP-GFP-LC3 Kit (P36239, Molecular Probes, Thermo Fisher Scientific, Bremen, Germany) that allows visualization of the maturation of the autophagosomes to the autolysosomes.

Techniques: Labeling, Fluorescence, Software

Journal: bioRxiv

Article Title: Extracellular vesicles promote autophagy in human microglia through lipid raft-dependent mechanisms

doi: 10.1101/2023.07.03.547488

Figure Lengend Snippet: A – Confocal images of labeled LC3B protein (red) in microglia cells in the presence and absence of pre-treatment with 10 μM of cilengitide for 2 hours (63× magnification immersion objective, scale bar = 100 μm). B – The mean fluorescence intensity of LC3B protein per cell were measured with Leica Application Suite X (LAS X) software, data shown represent the results of 15 fields of view for each experimental group from three independent biological experiments (n = 3), plotted as the mean ± SEM, results normalized to control. Statistical significance was analyzed by Kruskal–Wallis test followed by Dunn’s post-hoc test in GraphPad Prism 8.0.1 software (** p < 0.01; **** p < 0.0001)

Article Snippet: We used Premo™ Autophagy Tandem Sensor RFP-GFP-LC3 Kit (P36239, Molecular Probes, Thermo Fisher Scientific, Bremen, Germany) that allows visualization of the maturation of the autophagosomes to the autolysosomes.

Techniques: Labeling, Fluorescence, Software