Journal: Cell Communication and Signaling : CCS

Article Title: Extracellular vesicles derived from Enterococcus faecalis : inflammatory activation does not require internalization

doi: 10.1186/s12964-026-02926-9

Figure Lengend Snippet: Ef -EVs activate the NF-κB/AP-1 pathways in human reporter macrophages via TLR2 signaling. A dTHP1-XBlue cells were treated with Ef -EVs (100 − 50,000 EVs/cell). LPS (100 ng/mL) was used as a positive control. B dTHP1-XBlue cells were treated with EVs derived from clinical E. faecalis bloodstream isolates (1000-10,000 EVs/cell). LPS (100 ng/mL) and Pam 3 CSK 4 (100 ng/mL) were used as positive controls. C-E) dTHP1-XBlue cells were pretreated with increasing concentrations (from 0.1 to 5 µg/mL) of anti-hTLR2-IgA mAb (dark pink) or human IgA2 control mAb (light pink) for 1 h. Subsequently, cells were treated with either (C) EVs (7000 EVs/cell), (D) Pam 3 CSK 4 (TLR2 ligand, 1 ng/mL), or (E) LPS (TLR4 ligand, 1 ng/mL) in the presence of antibodies for 24 h. Cells treated with cell culture medium supplemented only with Ef -EVs (7000 EVs/cell) in (C, blue), Pam 3 CSK 4 (1 ng/mL) in (D, dark blue), or LPS (1 ng/mL) in (E, red) were used as positive activating controls while cells treated with cell culture medium supplemented alone ( C - E , light green) were used as negative controls. NF-κB/AP-1 activation was measured as the activity of secreted SEAP and expressed as a normalized value relative to the positive controls. Data are shown as means ± SD of three independent experiments ( N = 3, n ≥ 3) and analyzed either by Kruskal-Wallis test followed by Dunn’s multiple comparison post hoc test in (A and B) or by two-way analysis of variance (ANOVA) followed by Šídák multiple comparison post hoc test in ( C - E )

Article Snippet: NF-κB/AP-1 reporter HEK293 cells, endogenously expressing the human IL-1 receptor and stably co-transfected with the murine IL-1 receptor and a SEAP reporter gene under the control of the IFN-β minimal promoter fused to five NF-κB and five AP-1 binding sites (HEK-BlueTM IL-1R cells, cat. no. hkb-il1r, Invivogen), designed to detect bioactive human and murine IL-1α and IL-1β, were cultured in DMEM containing 4.5 g/L glucose, 2 mM L-glutamine, 10% heat-inactivated FCS, 100 μg/mL NormocinTM, 100 U/mL-100 μg/mL Pen-Strep, 100 μg/mL of ZeocinTM, 1 μg/mL of Puromycin, and 200 μg/mL Hygromycin B Gold at 37 °C with 5% CO2.

Techniques: Positive Control, Derivative Assay, Control, Cell Culture, Activation Assay, Activity Assay, Comparison

Journal: Cell Communication and Signaling : CCS

Article Title: Extracellular vesicles derived from Enterococcus faecalis : inflammatory activation does not require internalization

doi: 10.1186/s12964-026-02926-9

Figure Lengend Snippet: TLR2 controls EV-induced immune activation but does not function as an endocytic receptor for EV uptake. dTHP1-XBlue cells were pretreated with anti-hTLR2-IgA mAb (1 µg/mL) or human IgA2 control mAb (1 µg/mL) for 1 h. Cells were then treated with DiI-labeled Ef -EVs (7000 EVs/cell) in the presence of antibodies for 4 and 24 h. Cells incubated in cell culture medium alone were used as negative controls. Cells treated with cell culture medium supplemented with DiI-labeled Ef -EVs (7000 EVs/cell) were used as positive activating controls. For EV internalization assays, mean fluorescence intensity (B and C, upper panel) and EV-positive cells (B and C, lower panel) were quantified after 4 ( A and B ) and 24 h ( A and C ) of EV treatment by measuring fluorescence intensity associated with DiI-labeled Ef -EVs on the PE channel. NF-κB/AP-1 activation in D was measured after 24 h of EV incubation as the activity of secreted SEAP and expressed normalized to the positive controls. Quantitative results are presented as mean ± SD ( N = 3, n = 3) and were analyzed by Kruskal-Wallis test followed by Dunn’s multiple comparison post hoc test

Article Snippet: NF-κB/AP-1 reporter HEK293 cells, endogenously expressing the human IL-1 receptor and stably co-transfected with the murine IL-1 receptor and a SEAP reporter gene under the control of the IFN-β minimal promoter fused to five NF-κB and five AP-1 binding sites (HEK-BlueTM IL-1R cells, cat. no. hkb-il1r, Invivogen), designed to detect bioactive human and murine IL-1α and IL-1β, were cultured in DMEM containing 4.5 g/L glucose, 2 mM L-glutamine, 10% heat-inactivated FCS, 100 μg/mL NormocinTM, 100 U/mL-100 μg/mL Pen-Strep, 100 μg/mL of ZeocinTM, 1 μg/mL of Puromycin, and 200 μg/mL Hygromycin B Gold at 37 °C with 5% CO2.

Techniques: Activation Assay, Control, Labeling, Incubation, Cell Culture, Fluorescence, Activity Assay, Comparison

Journal: Cell Communication and Signaling : CCS

Article Title: Extracellular vesicles derived from Enterococcus faecalis : inflammatory activation does not require internalization

doi: 10.1186/s12964-026-02926-9

Figure Lengend Snippet: TLR2 engagement by Pam 3 CSK 4 -SUVs triggers inflammatory activation but does not enhance SUV internalization. A - C ) dTHP1-XBlue cells were treated with Pam 3 CSK 4 -SUVs (6 µM) containing increasing amounts of Pam 3 CSK 4 (from 0 to 0.4 mol% total SUV lipid composition) for 18 h. Cells incubated in cell culture medium alone were used as negative controls. Cells treated with cell culture medium containing Pam 3 CSK 4 -SUVs (6 µM, Pam 3 CSK 4 composition = 0.4 mol% total SUV lipid composition) were used as positive activating controls. D - F ) dTHP1-XBlue cells were pretreated with anti-hTLR2-IgA mAb (1 µg/mL) or human IgA2 control mAb (1 µg/mL) for 1 h. Cells were then treated with Pam 3 CSK 4 -SUVs (6 µM, Pam 3 CSK 4 composition = 0.04 mol% total SUV lipid composition) in the presence of antibodies for 18 h. Cells treated with cell culture medium containing Pam 3 CSK 4 -SUVs (6 µM, Pam 3 CSK 4 composition = 0.04 mol% total SUV lipid composition) were used as positive activating controls. For both experiments, cells incubated in cell culture medium alone were used as negative controls. Mean fluorescence intensity (B and E, upper panel) and SUV-positive cells (B and E, lower panel) were quantified after 18 h of treatment by measuring fluorescence intensity associated with SUVs on the APC channel. In C and F, NF-κB/AP-1 activation was measured after 18 h of SUV treatment as the activity of secreted SEAP and expressed normalized to the positive controls. Quantitative results are shown as mean ± SD ( N = 3, n = 3) and were analyzed by Kruskal-Wallis test followed by Dunn’s multiple comparison post hoc test

Article Snippet: NF-κB/AP-1 reporter HEK293 cells, endogenously expressing the human IL-1 receptor and stably co-transfected with the murine IL-1 receptor and a SEAP reporter gene under the control of the IFN-β minimal promoter fused to five NF-κB and five AP-1 binding sites (HEK-BlueTM IL-1R cells, cat. no. hkb-il1r, Invivogen), designed to detect bioactive human and murine IL-1α and IL-1β, were cultured in DMEM containing 4.5 g/L glucose, 2 mM L-glutamine, 10% heat-inactivated FCS, 100 μg/mL NormocinTM, 100 U/mL-100 μg/mL Pen-Strep, 100 μg/mL of ZeocinTM, 1 μg/mL of Puromycin, and 200 μg/mL Hygromycin B Gold at 37 °C with 5% CO2.

Techniques: Activation Assay, Incubation, Cell Culture, Control, Fluorescence, Activity Assay, Comparison

Journal: Cell Communication and Signaling : CCS

Article Title: Extracellular vesicles derived from Enterococcus faecalis : inflammatory activation does not require internalization

doi: 10.1186/s12964-026-02926-9

Figure Lengend Snippet: Routes of Ef -EV uptake. A and B ) dTHP1-XBlue cells were pretreated with pharmacological endocytosis inhibitors for 30 min. Afterward, cells were incubated with DiI-labeled Ef -EVs (7000 EVs/cell) in the presence of inhibitors for 4 h. Mean fluorescence intensity (B, upper panel) and EV-positive cells (B, lower panel) were quantified by measuring fluorescence intensity associated with DiI-labeled Ef -EVs on the PE channel. Cells treated by EVs dispersed in cell culture medium or medium containing DMSO (1% v/v) were used as positive uptake controls. C ) dTHP1-XBlue cells were pretreated with dynasore at 100 µM for 30 min. Afterward, cells were incubated with Ef -EVs (7000 EVs/cell) in the presence of dynasore at 100 µM for 24 h. Cells treated with cell culture medium supplemented only with Ef -EVs (7000 EVs/cell) were used as positive activating controls. NF-κB/AP-1 activation was measured as the activity of secreted SEAP and expressed as a normalized value relative to the positive control. Quantitative results are presented as mean ± SD of three independent experiments ( N = 3, n ≥ 3) and were analyzed by Kruskal-Wallis test followed by Dunn’s multiple comparison post hoc test

Article Snippet: NF-κB/AP-1 reporter HEK293 cells, endogenously expressing the human IL-1 receptor and stably co-transfected with the murine IL-1 receptor and a SEAP reporter gene under the control of the IFN-β minimal promoter fused to five NF-κB and five AP-1 binding sites (HEK-BlueTM IL-1R cells, cat. no. hkb-il1r, Invivogen), designed to detect bioactive human and murine IL-1α and IL-1β, were cultured in DMEM containing 4.5 g/L glucose, 2 mM L-glutamine, 10% heat-inactivated FCS, 100 μg/mL NormocinTM, 100 U/mL-100 μg/mL Pen-Strep, 100 μg/mL of ZeocinTM, 1 μg/mL of Puromycin, and 200 μg/mL Hygromycin B Gold at 37 °C with 5% CO2.

Techniques: Incubation, Labeling, Fluorescence, Cell Culture, Activation Assay, Activity Assay, Positive Control, Comparison

Journal: Frontiers in Pharmacology

Article Title: Activation of GLP-1R ameliorates alcohol withdrawal induced anxiety-like behavior by regulating neuronal mitochondrial quality control

doi: 10.3389/fphar.2026.1820128

Figure Lengend Snippet: Effects of CEE on anxiety-like behaviors and GLP-1R expression in mice. (A) Representative tracking plot from the OFT. (B) Number of entries in the center area (**p = 0.0015. n = 9 mice per group). (C) Time spent in the center area (**p = 0.0056. n = 9 mice per group). (D) Representative track plot of the EPM test. (E) Number of entries in the open arms (**p = 0.0070. n = 9 mice per group). (F) Time spent in the open arms during the EPM test (**p = 0.0056. n = 9 mice per group). (G,H) Protein expression of GLP-1R was detected by Western blot analysis (***p = 0.0002. n = 6 mice per group). (I) Representative images of Immunofluorescence staining of GLP-1R and NeuN with DAPI nuclear counterstaining in the PFC. *p = 0.0113. n = 3 mice per group). NeuN (green) was used to label mature neurons, and DAPI (blue) was used as a nuclear counterstain. GLP-1R (red) was predominantly co-localized with NeuN-positive neurons, indicating neuronal expression. Scale bar = 100 μm. (J) PFC dissection: bregma +1.69 mm. All data are presented as mean ± standard error of the mean (SEM). *p < 0.05, **p < 0.01, ***p < 0.001. Abbreviations: CEE, Chronic ethanol exposure; GLP-1R, Glucagon-like Peptide-1 receptor; OFT, Open Field Test; EPM, Elevated Plus Maze.

Article Snippet: Immunofluorescence staining was performed using primary antibodies against GLP-1R ( GB113881 , 1:5000, Servicebio) and NeuN (GB11138, 1:5000, Servicebio).

Techniques: Expressing, Western Blot, Immunofluorescence, Staining, Dissection

Journal: Frontiers in Pharmacology

Article Title: Activation of GLP-1R ameliorates alcohol withdrawal induced anxiety-like behavior by regulating neuronal mitochondrial quality control

doi: 10.3389/fphar.2026.1820128

Figure Lengend Snippet: Effects of Semaglutide on CEE-induced anxiety-like behaviors and GLP-1R expression. (A) Representative tracking plot from the OFT. (B) Number of entries in the center area (**p < 0.01. n = 19 mice per group). (C) Time spent in the center area (***p < 0.001. n = 19 mice per group). (D) Representative track plot of the EPM test. (E) Number of entries in the open arms (***p < 0.001. n = 19 mice per group). (F) Time spent in the open arms during the EPM test (*p < 0.05, ***p < 0.001. n = 19 mice per group). (G,H) Protein expression of GLP-1R was detected by Western blot analysis (**p < 0.01. n = 6 mice per group). (I) Representative confocal images of Immunofluorescence staining of GLP-1R and NeuN with DAPI nuclear counterstaining in the PFC. (*p < 0.05, **p < 0.01. n = 4 mice per group). NeuN (green) was used to label mature neurons, and DAPI (blue) was used as a nuclear counterstain. GLP-1R (red) was predominantly co-localized with NeuN-positive neurons, indicating neuronal expression. Scale bar = 100 μm. (J) PFC dissection: bregma +1.69 mm. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. Abbreviations: CEE, Chronic ethanol exposure; GLP-1R, Glucagon-like Peptide-1 receptor; OFT, Open Field Test; EPM, Elevated Plus Maze; Sema, Semaglutide.

Article Snippet: Immunofluorescence staining was performed using primary antibodies against GLP-1R ( GB113881 , 1:5000, Servicebio) and NeuN (GB11138, 1:5000, Servicebio).

Techniques: Expressing, Western Blot, Immunofluorescence, Staining, Dissection

Journal: Frontiers in Pharmacology

Article Title: Activation of GLP-1R ameliorates alcohol withdrawal induced anxiety-like behavior by regulating neuronal mitochondrial quality control

doi: 10.3389/fphar.2026.1820128

Figure Lengend Snippet: Effects of Semaglutide on mitochondrial damage in CEE mice. (A,B) Protein expression of mitochondrial GLP-1R was detected (*p < 0.05, ***p < 0.001. n = 6 mice per group). (C,D) Protein expression of mitochondrial CREB was detected by Western blot analysis (*p < 0.05, **p < 0.01. n = 6 mice per group). (E-J) Protein expression of mitochondrial OXPHOS was detected by Western blot analysis, including complex I (NDUFB8), complex II (SDHB), complex III (UQCRC2), complex IV (MTCO1), and complex V (ATP5A) (*p < 0.05, **p < 0.01. n = 6 mice per group). (K) Mitochondrial morphology of neurons in PFC of different groups. Scale bar = 2 μm. (L) Ratio of damaged mitochondria (Representative TEM images were used to calculate the percentage of damaged mitochondria. Mitochondrial damage was defined as the presence of mitochondrial swelling and/or cristae disruption, as described in the Materials and Methods.) (**p < 0.01, ***p < 0.001. n = 5 mice per group). Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. Abbreviations: GLP-1R, Glucagon-like Peptide-1 receptor; CREB, cAMP Response Element-Binding; OXPHOS, Oxidative phosphorylation; Sema, Semaglutide; NDUFB8, Ubiquinone oxidoreductase subunit B8; SDHB, Succinate dehydrogenase complex iron sulfur subunit B; UQCRC2, Ubiquinol-cytochrome c reductase core protein 2; MTCO1, Mitochondrially encoded cytochrome c oxidase I; vATP5A, ATP synthase F1 subunit alpha.

Article Snippet: Immunofluorescence staining was performed using primary antibodies against GLP-1R ( GB113881 , 1:5000, Servicebio) and NeuN (GB11138, 1:5000, Servicebio).

Techniques: Expressing, Western Blot, Disruption, Binding Assay, Phospho-proteomics

Journal: Frontiers in Pharmacology

Article Title: Activation of GLP-1R ameliorates alcohol withdrawal induced anxiety-like behavior by regulating neuronal mitochondrial quality control

doi: 10.3389/fphar.2026.1820128

Figure Lengend Snippet: Regulation of mitochondrial quality control by Semaglutide in CEE mice. (A) Mitochondrial quality control, including fission, fusion, and mitophagy. (B–D) Protein expression of mitochondrial FIS1, DRP1 and p-DRP1 were detected (*p < 0.05, **p < 0.01, ***p < 0.001. n = 6 mice per group). (E–G) Protein expression of mitochondrial MFN1 and MFN2 were detected (*p < 0.05. n = 6 mice per group). (H–J) Protein expressions of mitochondrial Parkin and Pink1 were detected *p < 0.05, ***p < 0.001. n = 6 mice per group). (K-M) Protein expressions of LC3B and P62 were detected (*p < 0.05, **p < 0.01. n = 6 mice per group). Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. Abbreviations: GLP-1R, Glucagon-like Peptide-1 receptor; Sema, Semaglutide; DRP1DRP1, Dynamin-related protein-1; FIS1, Fission, mitochondrial 1; MFN1, Mitofusin 1; MFN2, Mitofusin 2; Pink1, PTEN-induced kinase 1; Parkin, Parkin RBR E3 ubiquitin-protein ligase; LC3, Microtubule-associated protein 1A/1B-light chain 3.

Article Snippet: Immunofluorescence staining was performed using primary antibodies against GLP-1R ( GB113881 , 1:5000, Servicebio) and NeuN (GB11138, 1:5000, Servicebio).

Techniques: Control, Expressing, Ubiquitin Proteomics

Journal: Frontiers in Pharmacology

Article Title: Activation of GLP-1R ameliorates alcohol withdrawal induced anxiety-like behavior by regulating neuronal mitochondrial quality control

doi: 10.3389/fphar.2026.1820128

Figure Lengend Snippet: Synaptic morphology in PFC pyramidal neurons of CEE mice treated with Semaglutide. (A-C) Protein expressions of PSD95 and SYN were detected (*p < 0.05, ***p < 0.001. n = 6 mice per group). (D) Representative images of neurons in PFC labeled using Golgi staining. Scale bar = 20 μm. (E) Sholl analysis of PFC neurons revealing alterations in basal dendritic intersections at distinct distances from the soma. Two-way ANOVA with repeated measures revealed a significant effect of drug treatment and distance from the soma (*p < 0.05, **p < 0.01, ***p < 0.001. n = 4 mice per group). (F,G) Total dendritic length and dendritic branch number (**p < 0.01. n = 4 mice per group). (H) Representative images of dendritic spines by Golgi staining. (I) Spine density. Scale bar = 10 μm. (***P < 0.001. n = 4 mice per group). Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. Abbreviations: GLP-1R, Glucagon-like Peptide-1 receptor; Sema, Semaglutide; PSD95, Postsynaptic density protein 95; SYN, Synuclein.

Article Snippet: Immunofluorescence staining was performed using primary antibodies against GLP-1R ( GB113881 , 1:5000, Servicebio) and NeuN (GB11138, 1:5000, Servicebio).

Techniques: Labeling, Staining