Journal: Cell Communication and Signaling : CCS

Article Title: Oligodendroglia-derived extracellular vesicles activate autophagy via LC3B/BAG3 to protect against oxidative stress with an enhanced effect for HSPB8 enriched vesicles

doi: 10.1186/s12964-022-00863-x

Figure Lengend Snippet: OL-EVs and OL-HSPB8-EVs uptake and chaperone activity. A – D Quantification of cellular uptake of DiI-labelled OL-EVs and OL-HSPB8-EVs by flow cytometry in human microglia C20 and primary mixed neural cultures. The data presents the mean fluorescent intensity and are shown as mean ± SEM (n = 3 biological replicates per group. Green peaks show live cells stimulated with DiI-labelled EVs and/or activated with TNFα/IL1β while grey peaks show the non-stimulated, non-activated live cells E Relative qPCR expression of HSPB8 in human microglia C20 cell line stimulated with OL-EVs or OL-HSPB8-EVs either non-activated or TNFα/IL1β activated. Data were analyzed using stable reference genes, and the fold changes were plotted as the mean ± SEM and normalized to the non-stimulated control cells (n = 3 biological replicates per group). F – H Western blotting analysis of SQSTM1/P62 and ubiquitinated proteins in HeLa HSPB8-KO cells in the soluble (non-aggregated) and insoluble fraction (aggregated). The cells were either OL-EVs, OL-HSPB8-EVs stimulated or non-stimulated and exposed to a 90 min heat shock at 42 °C. Protein levels were calculated from two independent experiments and normalized to β-actin. Band intensities were determined by quantifying the mean pixel gray values using the ImageJ software. Data are presented as mean ± SEM. One-way ANOVA was used in GraphPad to determine statistical significance (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001)

Article Snippet: After 24 h, cells were activated with TNFα (100 ng/mL, Immunotools, 11343015) and IL1β (100 ng/mL, Immunotools, 11340015) to mimic inflammation.

Techniques: Activity Assay, Flow Cytometry, Expressing, Control, Western Blot, Software

Journal: Cell Communication and Signaling : CCS

Article Title: Oligodendroglia-derived extracellular vesicles activate autophagy via LC3B/BAG3 to protect against oxidative stress with an enhanced effect for HSPB8 enriched vesicles

doi: 10.1186/s12964-022-00863-x

Figure Lengend Snippet: OL-EVs and OL-HSPB8-EVs stimulation activate autophagy in human microglia and primary mixed neural cells. A , B Western blotting analysis of LC3B-II and BAG3 in OL-EVs and OL-HSPB8-EVs. Protein levels were calculated from two independent experiments and normalized to the EVs marker flotillin-1. Band intensities were determined by quantifying the mean pixel gray values using the ImageJ software. Data are presented as mean ± SEM. C , D Immunofluorescence staining of human microglial C20 cells stimulated with OL-EVs or OL-HSPB8-EVs. Cells were treated with chloroquine to inhibit lysosomal degradation. Rapamycin was used as a positive control. Representative image showing LC3B (green), the DAPI nucleus stain (blue), and merged images. CellProfiler was used to quantify the LC3B punctate structures (LC3B, mean fluorescence intensity) (n = 4 biological replicates, 15 + cells per experiment). Data are expressed as mean ± SEM, **** P < 0.0001. E – H Flow cytometry monitoring CYTO-ID autophagic flux in chloroquine treated human microglia C20 cell line and a primary mixed neural culture using the CYTO-ID green detection dye, selectively labeling autophagic vacuoles. Data are presented as mean fluorescent intensities ± SEM (n = 3 biological replicates per group), Green peaks represent TNFα/IL1β activated and/or EV stimulated cells, while the grey peaks show cells of non-activated and non-stimulated control cells. GraphPad was used to determine significance (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001). I , J Ultrastructural analysis of autophagic vesicles by transmission electron microscopy of C20 cells stimulated with OL-EVs or OL-HSPB8-EVs. Lysosomal degradation was blocked using chloroquine. Rapamycin was used as a positive control. The total number of autophagic vesicles per field of view were counted and expressed per cytoplasmic area covered (scale bar = 1 µm). Data are shown as mean ± SEM. GraphPad was used for statistical analysis by using one-way ANOVA (** P < 0.01)

Article Snippet: After 24 h, cells were activated with TNFα (100 ng/mL, Immunotools, 11343015) and IL1β (100 ng/mL, Immunotools, 11340015) to mimic inflammation.

Techniques: Western Blot, Marker, Software, Immunofluorescence, Staining, Positive Control, Fluorescence, Flow Cytometry, Labeling, Control, Transmission Assay, Electron Microscopy

Journal: Cell Communication and Signaling : CCS

Article Title: Oligodendroglia-derived extracellular vesicles activate autophagy via LC3B/BAG3 to protect against oxidative stress with an enhanced effect for HSPB8 enriched vesicles

doi: 10.1186/s12964-022-00863-x

Figure Lengend Snippet: OL-HSPB8-EVs reduce reactive oxygen species upon PMA stimulation. A – D Flow cytometry monitoring of ROS activity assay in human microglia C20 cell line ( A , B ) and a primary mixed neural culture ( C , D ) using the DCFH-DA detection dye. Data are presented as mean fluorescent intensities ± SEM (n = 3 biological replicates per group). Graphpad was used to determine significance by using a one-way ANOVA statistical analysis (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001). E Relative qPCR expression of ROS-related genes SOD1 and SOD2 in human C20 cells stimulated with OL-EV or OL-HSPB8-EV either non-activated or TNFα/IL1β activated. As a positive control, the ROS inducer PMA was used. Values are represented as the mean ± SEM of three independent biological replicates. Data were normalized to non-stimulated equals 1. One-way ANOVA was used to determine statistical significance where P < 0.05 was considered statistically significant

Article Snippet: After 24 h, cells were activated with TNFα (100 ng/mL, Immunotools, 11343015) and IL1β (100 ng/mL, Immunotools, 11340015) to mimic inflammation.

Techniques: Flow Cytometry, Activity Assay, Expressing, Positive Control

Journal: Cell Communication and Signaling : CCS

Article Title: Oligodendroglia-derived extracellular vesicles activate autophagy via LC3B/BAG3 to protect against oxidative stress with an enhanced effect for HSPB8 enriched vesicles

doi: 10.1186/s12964-022-00863-x

Figure Lengend Snippet: OL-HSPB8-EVs do not promote cell death in C20 cell line and primary mixed neural cultures. A – F Flow cytometry quantification of early apoptosis by using the JC1 dye to detect the mitochondrial membrane potential ( A – D ) and by using Annexin V to measure phosphatidylserine exposure E , F in the human microglial C20 cell line and a primary mixed neural culture stimulated with OL-EVs or OL-HSPB8-EVs. Cells were either activated with TNFα/IL1β or non-activated. The positive control was stimulated with PMA or staurosporine dependent for each experiment. Data are presented as mean percentage ± SEM (n = 3 biological replicates per group). Graphpad was used to determine significance by using a one-way ANOVA statistical analysis (* P < 0.05, ** P < 0.01). G Relative qPCR expression of the apoptosis-related genes Caspase1, Caspase8, FAS, IL1β, TRAIL, FADD, and VEGF in human C20 cells stimulated with OL-EVs or OL-HSPB8-EVs either non-activated, or TNFα/IL1β activated. As a positive control, the apoptosis inducer staurosporine was used. Values are represented as the mean ± SEM of three independent biological replicates. Data were normalized to non-stimulated equals 1. One-way ANOVA was used to determine statistical significance were P < 0.05 was considered as statistically significant (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001)

Article Snippet: After 24 h, cells were activated with TNFα (100 ng/mL, Immunotools, 11343015) and IL1β (100 ng/mL, Immunotools, 11340015) to mimic inflammation.

Techniques: Flow Cytometry, Membrane, Positive Control, Expressing

Journal: Journal for Immunotherapy of Cancer

Article Title: Tumor cell SYK expression modulates the tumor immune microenvironment composition in human cancer via TNF-α dependent signaling

doi: 10.1136/jitc-2022-005113

Figure Lengend Snippet: TNFα induces SYK expression in lung cancer cells and SYK mediates proinflammatory responses. (A–D) SYK protein expression measured by flow cytometry in human lung cancer cell lines A549 (KRAS mutated) and H1975 (EGFR mutated) in vitro stimulated with human recombinant TNFα or IFNγ for 24 hours. Panels A and C show histograms displaying the difference in the signal intensity obtained using isotype control staining (gray line) and anti-SYK PE-conjugated antibody (red line). (E–H) A549 and H1975 cell lines were left in control condition (medium) or treated with TNFα, TAK-659 or TAK-659 plus TNFα for 24 hours. After that, cells were recovered, stained with antibodies against SYK-PE (E and F), HLA-ABC-FITC (G and H) and analyzed by flow cytometry. (I–L) A549 and H1975 cells lines were treated with TAK659, TNFα alone or the combination for 24 hours. After that, samples were digested and total RNA was obtained using a high pure RNA isolation kit and transcript were analyzed using the NanoString platform targeted mRNA expression of 770 immune-related transcripts. The number of cases included in each analysis is indicated within each bar. *P<0.05; **p<0.01. SYK, spleen tyrosine kinase.

Article Snippet: Cells were preincubated for 24 hours in serum-free RPMI-1640 medium and then exposed to recombinant TNFα (20-50-100 ng/mL) or IFNγ (20-40-80 ng/mL) (R&D System, USA) for 24 hours in serum-free RPMI-1640.

Techniques: Expressing, Flow Cytometry, In Vitro, Recombinant, Staining, Isolation