Journal: PLoS ONE

Article Title: Combined Effect of Insulin-Like Growth Factor-1 and CC Chemokine Ligand 2 on Angiogenic Events in Endothelial Cells

doi: 10.1371/journal.pone.0121249

Figure Lengend Snippet: tEnd.1 cells were treated with IGF-1 (A) or CCL2 (B) at concentrations of 5, 10, 50, or 100 ng/mL, and cell viability was determined by cell counting using a hemocytometer or MTT assay, respectively. (C) Flow cytometry results are presented as histograms of the average percentage of cells that expressed IGF-1R and CCR2 receptors (gray) and immunoglobulin control. Values and bars are represented as the mean ± SEM (n = 4/group). Results were analyzed by one-way ANOVA followed by Bonferroni’s post-test. Significant values compared to the control group: p < 0.05 (*) or p < 0.0001(***); significant value compared to control group and the other treatments: p < 0.0001 (#).

Article Snippet: Cells were then treated with recombinant mouse CCL2/JE/MCP-1 (CCL2) (R&D Systems, Minneapolis, MN, USA) at concentrations of 5, 10, 50, and 100 ng/mL for 24 h. After treatment, cells were incubated with 5 mg/mL of tetrazolium salt (MTT) (Sigma-Aldrich) diluted in RPMI 1640 with 2% FBS.

Techniques: Cell Counting, MTT Assay, Flow Cytometry, Control

Journal: PLoS ONE

Article Title: Combined Effect of Insulin-Like Growth Factor-1 and CC Chemokine Ligand 2 on Angiogenic Events in Endothelial Cells

doi: 10.1371/journal.pone.0121249

Figure Lengend Snippet: tEnd.1 cells were treated with IGF-1 (100 ng/mL), CCL2 (10 ng/mL), or a combination of both for 24 h and analyzed by fluorescence microscopy. ( A ) Photomicrographs show the expression of FN ascertained by immunofluorescence and fluorescence microscopy analysis. Magnification: 400× ( B ) Bars correspond to the quantitative analysis of FN expression in tEnd.1 cells in selected microscopic fields (n = 5/group). The results are expressed in pixels/μm 2 . ( C ) Flow cytometry results are presented as histograms of the average percentage of cells that expressed CD49e/VLA-5 and CD44 receptors for FN (gray) and immunoglobulin control. Values and bars are represented as the mean ± SEM (n = 5/group). Results were analyzed by one-way ANOVA followed by Bonferroni’s post-test. Significant values compared to control group: p < 0.0001 (***); significant values compared to control group and single treatments: p < 0.0001 (#).

Article Snippet: Cells were then treated with recombinant mouse CCL2/JE/MCP-1 (CCL2) (R&D Systems, Minneapolis, MN, USA) at concentrations of 5, 10, 50, and 100 ng/mL for 24 h. After treatment, cells were incubated with 5 mg/mL of tetrazolium salt (MTT) (Sigma-Aldrich) diluted in RPMI 1640 with 2% FBS.

Techniques: Fluorescence, Microscopy, Expressing, Immunofluorescence, Flow Cytometry, Control

Journal: PLoS ONE

Article Title: Combined Effect of Insulin-Like Growth Factor-1 and CC Chemokine Ligand 2 on Angiogenic Events in Endothelial Cells

doi: 10.1371/journal.pone.0121249

Figure Lengend Snippet: (A) tEnd.1 cells treated with IGF-1 (100 ng/mL), CCL2 (10 ng/mL), or a combination of both for 24 h on BSA or FN coating were stained with Alexa 488-phalloidin and analyzed by confocal microscopy with a 63× objective. ( B ) tEnd.1 cells were allowed to adhere on BSA- or FN-coated surfaces for 1 h after stimulation with IGF-1, CCL2, or IGF-1/CCL2 for 24 h. ( C ) tEnd.1 cells were allowed to migrate through transwell chambers coated with BSA or FN after chemotactic stimulation with IGF-1, CCL2, or IGF-1/CCL2 for 6 h. Photomicrographs demonstrate cells invading through the transwell membrane. Giemsa staining. Scale bar = 10 μm. ( D ) Bars represent the number of migrating cells in a transwell system. Data are represented as mean ± SEM (n = 5/group). Results were analyzed by two-way ANOVA followed by Bonferroni’s post-test. Significant values compared to control group: p < 0.05 (*), p < 0.01 (**), or p < 0.0001 (***); significant values compared to control group and the IGF-1 treatment: p < 0.05 (#); and significant values compared to control group and single treatments: p < 0.01 (+).

Article Snippet: Cells were then treated with recombinant mouse CCL2/JE/MCP-1 (CCL2) (R&D Systems, Minneapolis, MN, USA) at concentrations of 5, 10, 50, and 100 ng/mL for 24 h. After treatment, cells were incubated with 5 mg/mL of tetrazolium salt (MTT) (Sigma-Aldrich) diluted in RPMI 1640 with 2% FBS.

Techniques: Staining, Confocal Microscopy, Membrane, Control

Journal: PLoS ONE

Article Title: Combined Effect of Insulin-Like Growth Factor-1 and CC Chemokine Ligand 2 on Angiogenic Events in Endothelial Cells

doi: 10.1371/journal.pone.0121249

Figure Lengend Snippet: ( A ) tEnd.1 cells were treated with IGF-1 (100 ng/mL), CCL2 (10 ng/mL), or a combination of both for 24 h and analyzed by optical microscopy. Photomicrographs show intracellular lumina in tEnd.1 cells, indicated by arrows. Giemsa staining. Scale bar = 10 μm. ( B ) tEnd.1 cells were treated with IGF-1, CCL2, or IGF-1/CCL2 for 8 days on BSA or FN coating and analyzed by optical microscopy. Photomicrographs demonstrate capillary-like structures, indicated by asterisks. Giemsa staining. Scale bar = 10 μm. ( C ) Number of capillary-like structures. ( D ) Luminal area of capillary-like structures. Bars represent the mean ± SEM (n = 6/group). Results were analyzed by two-way ANOVA followed by Bonferroni’s post-test. Significant compared with control, p < 0.05 (*), p < 0.01 (**), or p < 0.001 (***).

Article Snippet: Cells were then treated with recombinant mouse CCL2/JE/MCP-1 (CCL2) (R&D Systems, Minneapolis, MN, USA) at concentrations of 5, 10, 50, and 100 ng/mL for 24 h. After treatment, cells were incubated with 5 mg/mL of tetrazolium salt (MTT) (Sigma-Aldrich) diluted in RPMI 1640 with 2% FBS.

Techniques: Microscopy, Staining, Control

Journal: International journal of biological sciences

Article Title: PD-L1 promotes GSDMD-mediated NET release by maintaining the transcriptional activity of Stat3 in sepsis-associated encephalopathy.

doi: 10.7150/ijbs.79913

Figure Lengend Snippet: Figure 1. Neutrophils accumulate in the hippocampus and contribute to the progression of SAE. (A and B) Representative immunoblots and quantification of Ly6G levels in the hippocampus at 24 hours after operation. (C and D) Representative FACS plots and quantification of neutrophils (Ly6G+CD11b+) measured by flow cytometry in blood at 24 hours after operation. (E and F) Freezing to context and freezing to tone examined at 24 hours after operation. (G) The BBB permeability of hippocampus evaluated by Evans blue extravasation at 24 hours after operation. (H) Representative TUNEL (green) and DAPI (blue) immunofluorescence staining in the hippocampus. Scale bar indicates 20 μm. Higher magnification images are shown at the right row of figures-scale bar indicates 10 μm. (I) The quantitative results of the percentage of TUNEL-positive area in the total area of the image (whole microscopic field) in the hippocampus. (J) Representative IBA-1 (red) and DAPI (blue) immunofluorescence staining in the hippocampus. Scale bar indicates 20 μm. Higher magnification images are shown at the right row of figures-scale bar indicates 10 μm. (K) The quantitative results of the percentage of IBA-1 positive area in the total area of the image (whole microscopic field) in the hippocampus. The values are presented as mean ± SD (n=6 for Fig. B, D, G, I and K; n=8 for Fig. E and F; **P<0.01, ***P<0.001, ****P<0.0001, ns=not significant, one-way analysis of variance).

Article Snippet: Mice blood neutrophils were isolated by positive selection magnetic cell separation (MACS) using the Neutrophil Isolation Kit (130-097-658, Miltenyi Biotec, Bergisch Gladbach, Germany) according to instructions.

Techniques: Western Blot, Flow Cytometry, Permeability, TUNEL Assay, Immunofluorescence, Staining

Journal: International journal of biological sciences

Article Title: PD-L1 promotes GSDMD-mediated NET release by maintaining the transcriptional activity of Stat3 in sepsis-associated encephalopathy.

doi: 10.7150/ijbs.79913

Figure Lengend Snippet: Figure 2. Sepsis induces neutrophil releasing NETs and NETs digestion with DNase I attenuates the development of SAE. (A) Representative immunofluorescence images of isolated peripheral blood neutrophils at 24 hours after operation. Neutrophils are stained with SYTOX Orange (red) and Cit-H3 (green). Arrows indicate NETs. Scale bar indicates 10 μm. (B) Quantification of the percentage of Cit-H3-positive neutrophils. (C) Levels of plasma cfDNA are measured at 24 hours after operation. (D and E) Representative immunoblots of NETs appearance (D) and quantification of the Cit-H3 levels (E) in the hippocampus at 24 hours after operation. (F) Representative immunofluorescence images of Cit-H3 (green) and MPO (red) staining with blue DAPI nuclear staining in hippocampus. Neutrophils express MPO (red) and NET forming neutrophils also express Cit-H3 (green). Cyan fluorescence represents the colocalization of Cit-H3 with DNA. The white arrows point to neutrophils with NETs and the red arrows to neutrophils without NETs. Scale bar indicates 20 μm. Higher magnification images are shown at the right row of figures-scale bar indicates 10 μm. (G) Total NETs score of each group. (H and I) Freezing to context and freezing to tone examined at 24 hours after operation. (J) The BBB permeability of hippocampus evaluated by Evans blue extravasation at 24 hours after operation. (K) Representative TUNEL (green) and DAPI (blue) immunofluorescence staining in the hippocampus. Scale bar indicates 20 μm. Higher magnification images are shown at the right row of figures-scale bar indicates 10 μm. (L) The quantitative results of the percentage of TUNEL positive area in the total area of the image (whole microscopic field) in the hippocampus. (M) Representative IBA-1 (red) and DAPI (blue) immunofluorescence staining in the hippocampus. Scale bar indicates 20 μm. Higher magnification images are shown at the right row of figures, and the scale bar indicates 10 μm. (N) The quantitative results of the percentage of IBA-1 positive area in the total area of the image (whole microscopic field) in the hippocampus. The values are presented as mean ± SD (n=6; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, ns=not significant, one-way analysis of variance).

Article Snippet: Mice blood neutrophils were isolated by positive selection magnetic cell separation (MACS) using the Neutrophil Isolation Kit (130-097-658, Miltenyi Biotec, Bergisch Gladbach, Germany) according to instructions.

Techniques: Immunofluorescence, Isolation, Staining, Clinical Proteomics, Western Blot, Fluorescence, Permeability, TUNEL Assay

Journal: International journal of biological sciences

Article Title: PD-L1 promotes GSDMD-mediated NET release by maintaining the transcriptional activity of Stat3 in sepsis-associated encephalopathy.

doi: 10.7150/ijbs.79913

Figure Lengend Snippet: Figure 3. GSDMD regulates NET release and GSDMD deficiency in neutrophils attenuates the progression of SAE. (A) Representative immunofluorescence images of isolated peripheral blood neutrophils at 24 hours after operation. Neutrophils are stained with SYTOX Orange (red) and Cit-H3 (green). Arrows indicate NETs. Scale bar indicates 10 μm. (B) Quantification of the percentage of Cit-H3-positive neutrophils. (C) Levels of plasma cfDNA are measured at 24 hours after operation. (D and E) Representative immunoblots of NETs appearance (D) and quantification of the Cit-H3 levels (E) in the hippocampus at 24 hours after operation. (F) Representative immunofluorescence images of Cit-H3 (green) and MPO (red) staining with blue DAPI nuclear staining in hippocampus. Neutrophils express MPO (red) and NET forming neutrophils also express Cit-H3 (green). Cyan fluorescence represents the colocalization of Cit-H3 with DNA. The white arrows point to neutrophils with NETs and the red arrows to neutrophils without NETs. Scale bar indicates 20 μm. Higher magnification images are shown at the right row of figures-scale bar indicates 10 μm. (G) Total NETs score of each group. (H and I) Freezing to context and freezing to tone examined at 24 hours after operation. (J) The BBB permeability of hippocampus evaluated by Evans blue extravasation at 24 hours after operation. (K) Representative TUNEL (green) and DAPI (blue) immunofluorescence staining in the hippocampus. Scale bar indicates 20 μm. Higher magnification images are shown at the right row of figures-scale bar indicates 10 μm. (L) The quantitative results of the percentage of TUNEL positive area in the total area of the image (whole microscopic field) in the hippocampus. (M) Representative IBA-1 (red) and DAPI (blue) immunofluorescence staining in the hippocampus. Scale bar indicates 20 μm. Higher magnification images are shown at the right row of figures-scale bar indicates 10 μm. (N) The quantitative results of the percentage of IBA-1 positive area in the total area of the image (whole microscopic field) in the hippocampus. The values are presented as mean ± SD (n=6; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, ns=not significant, one-way analysis of variance).

Article Snippet: Mice blood neutrophils were isolated by positive selection magnetic cell separation (MACS) using the Neutrophil Isolation Kit (130-097-658, Miltenyi Biotec, Bergisch Gladbach, Germany) according to instructions.

Techniques: Immunofluorescence, Isolation, Staining, Clinical Proteomics, Western Blot, Fluorescence, Permeability, TUNEL Assay

Journal: International journal of biological sciences

Article Title: PD-L1 promotes GSDMD-mediated NET release by maintaining the transcriptional activity of Stat3 in sepsis-associated encephalopathy.

doi: 10.7150/ijbs.79913

Figure Lengend Snippet: Figure 4. PD-L1 can regulate GSDMD expression in septic neutrophils. (A to C) Representative immunoblots and quantification of PD-L1 and GSDMD levels in the neutrophils from healthy subjects and septic patients. (D to F) Representative immunoblots and quantification of PD-L1 and GSDMD levels in the neutrophils from septic patients at 21 hours after PD-L1 siRNA treatment. (G) The GSDMD mRNA levels of neutrophils from septic patients at 21 hours after PD-L1 siRNA treatment. (H) Representative immunofluorescence images of isolated peripheral blood neutrophils from healthy subjects and septic patients. Neutrophils are stained with SYTOX Orange (red) and Cit-H3 (green). Arrows indicate NETs. Scale bar indicates 10 μm. (I) Quantification of the percentage of Cit-H3-positive neutrophils. (J) cfDNA levels of supernatant of cultured neutrophils are measured at 21 hours after PD-L1 siRNA treatment. (K) The nucleus is extracted from neutrophils from healthy subjects and septic patients. Representative immunoblot of PD-L1 in neutrophil nucleus. (L) Representative images of neutrophils PD-L1 from healthy subjects and septic patients by confocal microscopy. Neutrophils are stained with PD-L1 (green) and DAPI (blue). Scale bar indicates 10 μm. Higher magnification images are shown at the right row of figures-scale bar indicates 5 μm. The values are presented as mean ± SD (n=6; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, ns=not significant, 2-tailed Student’s t test for 4B, 4C, 4E, and 4F; one-way analysis of variance for 4G, 4I, 4J).

Article Snippet: Mice blood neutrophils were isolated by positive selection magnetic cell separation (MACS) using the Neutrophil Isolation Kit (130-097-658, Miltenyi Biotec, Bergisch Gladbach, Germany) according to instructions.

Techniques: Expressing, Western Blot, Immunofluorescence, Isolation, Staining, Cell Culture, Confocal Microscopy

Journal: International journal of biological sciences

Article Title: PD-L1 promotes GSDMD-mediated NET release by maintaining the transcriptional activity of Stat3 in sepsis-associated encephalopathy.

doi: 10.7150/ijbs.79913

Figure Lengend Snippet: Figure 5. nPD-L1 forms a complex with p-Y705-Stat3 to transcriptionally activate GSDMD expression in neutrophils from septic patients. (A and B) Representative immunoblots and quantification of p-Y705-Stat3 level in the neutrophils from healthy subjects and sepsis patients. (C) Immunoprecipitation (IP) and western blot analysis of the PD-L1/p-Y705-Stat3 interaction in neutrophils from healthy subjects and septic patients. (D) Representative images of neutrophils PD-L1/p-Y705-Stat3 interaction from healthy subjects and septic patients by confocal microscopy. Neutrophils are stained with p-Y705-Stat3 (green), PD-L1 (red) and DAPI (blue). Scale bar indicates 10 μm. Higher magnification images are shown at the right row of figures, and the scale bar indicates 5 μm. (E and F) The nucleus is extracted from septic neutrophils at 24 hours after treating with DMSO or inhibitor HO-3867 (20 μM). Representative immunoblots and quantification of PD-L1 level in neutrophil nucleus. (G and H) Representative immunoblots and quantification of GSDMD level in the neutrophils from septic patients at 24 hours after treating with DMSO or inhibitor HO-3867 (20 μM). (I) The GSDMD mRNA levels of neutrophils from septic patients at 24 hours after treating with DMSO or inhibitor HO-3867 (20 μM). (J) Sequential ChIP-PCR analysis of the interactions between p-Y705-Stat3 and the GSDMD promoter in septic neutrophils. The values are presented as mean ± SD (n=6; **P<0.01, ****P<0.0001, ns=not significant, 2-tailed Student’s t test for 5B; one-way analysis of variance for 5F, 5H, and 5I).

Article Snippet: Mice blood neutrophils were isolated by positive selection magnetic cell separation (MACS) using the Neutrophil Isolation Kit (130-097-658, Miltenyi Biotec, Bergisch Gladbach, Germany) according to instructions.

Techniques: Expressing, Western Blot, Immunoprecipitation, Confocal Microscopy, Staining

Journal: International journal of biological sciences

Article Title: PD-L1 promotes GSDMD-mediated NET release by maintaining the transcriptional activity of Stat3 in sepsis-associated encephalopathy.

doi: 10.7150/ijbs.79913

Figure Lengend Snippet: Figure 6. Genetic deletion of neutrophil PD-L1 reduces the expression of GSDMD in neutrophils in vivo and attenuates the release of NETs in CLP mice. (A and B) Representative FACS plots and quantification of GSDMD+ neutrophils measured by flow cytometry in blood at 24 hours after operation. (C) Representative immunofluorescence images of isolated peripheral blood neutrophils at 24 hours after operation. Neutrophils were stained with GSDMD (green) and DAPI (blue). Scale bar indicates 20 μm. Arrows indicate GSDMD+ neutrophils. (D) Quantification of the percentage of GSDMD-positive neutrophils. (E) Representative immunofluorescence images of isolated peripheral blood neutrophils at 24 hours after operation. Neutrophils are stained with SYTOX Orange (red) and Cit-H3 (green). Arrows indicate NETs. Scale bar indicates 10 μm. (F) Quantification of the percentage of Cit-H3-positive neutrophils. (G) Levels of plasma cfDNA are measured at 24 hours after sham or CLP surgery. (H and I) Representative immunoblots of NETs appearance (H) and quantification of the Cit-H3 levels (I) in the hippocampus at 24 hours after operation. (J) Representative immunofluorescence images of Cit-H3 (green) and MPO (red) staining with blue DAPI nuclear staining in hippocampus. Neutrophils express MPO (red) and NET forming neutrophils also express Cit-H3 (green). Cyan fluorescence represents the colocalization of Cit-H3 with DNA. The white arrows point to neutrophils with NETs and the red arrows to neutrophils without NETs. The scale bar indicates 20 μm. Higher magnification images are shown at the right row of figures-scale bar indicates 10 μm. (K) Total NETs score of each group. The values are presented as mean ± SD (n=6; ****P<0.0001, ns=not significant, one-way analysis of variance).

Article Snippet: Mice blood neutrophils were isolated by positive selection magnetic cell separation (MACS) using the Neutrophil Isolation Kit (130-097-658, Miltenyi Biotec, Bergisch Gladbach, Germany) according to instructions.

Techniques: Expressing, In Vivo, Flow Cytometry, Immunofluorescence, Isolation, Staining, Clinical Proteomics, Western Blot, Fluorescence

Journal: International journal of biological sciences

Article Title: PD-L1 promotes GSDMD-mediated NET release by maintaining the transcriptional activity of Stat3 in sepsis-associated encephalopathy.

doi: 10.7150/ijbs.79913

Figure Lengend Snippet: Figure 7. Genetic knockout of Neutrophil PD-L1 attenuates the progression of SAE. (A and B) Freezing to context and freezing to tone examined at 24 hours after operation. (C) The BBB permeability of hippocampus evaluated by Evans blue extravasation at 24 hours after operation. (D) Representative TUNEL (green) and DAPI (blue) immunofluorescence staining in the hippocampus. Scale bar indicates 20 μm. Higher magnification images are shown at the right row of figures-scale bar indicates 10 μm. (E) The quantitative results of the percentage of TUNEL positive area in the total area of the image (whole microscopic field) in the hippocampus. (F) Representative IBA-1 (red) and DAPI (blue) immunofluorescence staining in the hippocampus. Scale bar indicates 20 μm. Higher magnification images are shown at the right row of figures-scale bar indicates 10 μm. (G) The quantitative results of the percentage of IBA-1 positive area in the total area of the image (whole microscopic field) in the hippocampus. (H) Schematic illustration of the main findings: Sepsis-induced NETosis contributes to hippocampus-dependent memory impairment, and increases BBB permeability, neuronal apoptosis, and microglia activation in the hippocampus region. The NET release is promoted by the cleavage of GSDMD, which is transcriptionally regulated by the nuclear translocation of a PD-L1/p-Y705-Stat3 complex. Together, PD-L1/Stat3/GSDMD is essential for NET production and development of sepsis-associated encephalopathy. The values are presented as mean ± SD (n=6; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, one-way analysis of variance).

Article Snippet: Mice blood neutrophils were isolated by positive selection magnetic cell separation (MACS) using the Neutrophil Isolation Kit (130-097-658, Miltenyi Biotec, Bergisch Gladbach, Germany) according to instructions.

Techniques: Knock-Out, Permeability, TUNEL Assay, Immunofluorescence, Staining, Activation Assay, Translocation Assay

Journal: eLife

Article Title: Clearance of senescent decidual cells by uterine natural killer cells in cycling human endometrium

doi: 10.7554/eLife.31274

Figure Lengend Snippet: ( A ) Representative SAβG staining in undifferentiated EnSCs (Day 0) or cells decidualized for the indicated time points with 8-bromo-cAMP and MPA. Scale bar = 100 µm. ( B ) SAβG activity, expressed in fluorescence intensity units (FIU), in undifferentiated EnSCs (day 0) or cells decidualized for the indicated time points. ( C ) Representative Western blot analysis of p53, p16, LMNB1, HMGB2, mH2A, H3K9me3 and H.H1 levels in undifferentiated EnSCs and cells decidualized for the indicated time points. β-actin served as a loading control. ( D ) Left panel: representative immunofluorescence staining for p16 expression in undifferentiated cells and cells decidualized for 8 days. Nuclei were counterstained with DAPI. Scale bar = 50 µm. Right panel: percentage of p16 + cells. ( E ) Left panel: representative confocal microscopy images of undifferentiated (Day 0) or decidualized (Day 8) EnSCs immune-probed for LMNB1, mH2A, H3K9me3 and H.H1. Scale bar = 10 µm. Right panel: nuclear size of undifferentiated EnSCs (n = 48) and of cells first decidualized for 8 days with 8-br-cAMP and MPA (C + M) (n = 48) was measured in three primary cultures. ( F ) Secretion of IL-8, GROα, and IL-6 was measured in the supernatant of primary EnSCs collected every 48 hr over an 8 day decidualization time-course. Data are mean ±SEM of 3 biological replicates unless stated otherwise. ** p < 0.01, *** p < 0.001. Different letters above the error bars indicate that those groups are significantly different from each other at p<0.05. 10.7554/eLife.31274.004 Figure 1—source data 1. Decidualization induces acute senescence in a subpopulation of EnSCs.

Article Snippet: ELISAs were performed exactly as per manufacturer’s instructions (DuoSet ELISA kits for IL-8 (D8000C), IL-15 (D1500), GROα (DY275) and IL-6 (DY206); Bio-Techne, Abingdon, UK).

Techniques: Staining, Activity Assay, Fluorescence, Western Blot, Immunofluorescence, Expressing, Confocal Microscopy

Journal: eLife

Article Title: Clearance of senescent decidual cells by uterine natural killer cells in cycling human endometrium

doi: 10.7554/eLife.31274

Figure Lengend Snippet: ( A ) SAβG activity in EnSCs either undifferentiated, or decidualized for 8 days with 8-bromo-cAMP, MPA, or a combination. ( B ) Top left panel: FOXO1 mRNA levels in undifferentiated EnSCs and cells treated with 8-br-cAMP and MPA (C + M) following transfection with non-targeting (NT) or FOXO1 siRNA. Other panels: Secretion of IL-8, IL-6 and GROα was measured following FOXO1 knockdown in the supernatant of primary EnSCs every 48 hr over an 8 day decidualization time-course. ( C ) SAβG activity in EnSCs following transfection with NT or FOXO1 siRNA. The cultures either remain untreated or decidualized for 8 days. ( D ) SAβG activity in undifferentiated EnSCs treated for 8 days with increasing concentrations of recombinant IL-8 and in cells decidualized for 8 days in the presence of increasing concentrations of the CXCR2 antagonist, SB265610. ( E ) SAβG activity in EnSCs following transfection with IL-8 siRNA. The cultures either remain untreated or decidualized for 8 days. ( F ) PRL and IGFBP1 transcript levels in EnSCs following transfection with IL-8 siRNA. The cultures either remain untreated or decidualized for 8 days. ( G ) PRL and IGFBP1 expression in undifferentiated EnSCs, cells decidualized for 8 days, and upon withdrawal of 8-br-cAMP and MPA (C + M) for the indicated days. ( H ) Left panel: SAβG activity in undifferentiated EnSCs, cells decidualized for 8 days, and following withdrawal of C + M for the indicated days. Right panel: representative Western blot analysis of p53, p16, LMNB1 and HMGB2 levels in undifferentiated EnSCs, cells decidualized for 8 days, and following withdrawal of C + M for the indicated days. β-actin served as a loading control. Data are mean ±SEM of 3 biological replicates unless stated otherwise. *p < 0.05, **p < 0.01 and ***p < 0.005. Different letters above the error bars indicate that those groups are significantly different from each other at p<0.05. 10.7554/eLife.31274.009 Figure 3—source data 1. A FOXO1/IL-8 axis drives EnSC differentiation and senescence.

Article Snippet: ELISAs were performed exactly as per manufacturer’s instructions (DuoSet ELISA kits for IL-8 (D8000C), IL-15 (D1500), GROα (DY275) and IL-6 (DY206); Bio-Techne, Abingdon, UK).

Techniques: Activity Assay, Transfection, Recombinant, Expressing, Western Blot

Journal: eLife

Article Title: Clearance of senescent decidual cells by uterine natural killer cells in cycling human endometrium

doi: 10.7554/eLife.31274

Figure Lengend Snippet: ( A ) PRL and IGFBP1 transcript levels in EnSCs following transfection with FOXO1 siRNA. The cultures either remained untreated or were decidualized for 8 days. ( B ) Left panel: SAβG staining in undifferentiated EnSCs that remained untreated (control) or were incubated with recombinant IL-8 (30 μM) for 8 days. SAβG staining was also performed in parallel cultures decidualized with 8-bromo-cAMP and MPA (C + M) in the absence or presence of the CXCR2 antagonist SB265610 (10 μM). Right panel: IL-8 concentration in conditioned media from decidualized EnSCs following siRNA-mediated CXCL8 (IL-8) knockdown. ( C ) SAβG staining (left panel) and activity (right panel) in undifferentiated (day 0) and decidualized (day 8) EnSCs in the presence of the mTOR inhibitor rapamycin. FIU: fluorescence intensity units. ( D ) PRL and IGFPB1 transcripts in undifferentiated EnSCs and cells decidualized for 8 days in the presence or absence of rapamycin (100 nM). All data are mean ±SEM of 3 biological replicates. Different letters above the error bars indicate that those groups are significantly different from each other at p<0.05. Scale bars = 100 μm.

Article Snippet: ELISAs were performed exactly as per manufacturer’s instructions (DuoSet ELISA kits for IL-8 (D8000C), IL-15 (D1500), GROα (DY275) and IL-6 (DY206); Bio-Techne, Abingdon, UK).

Techniques: Transfection, Staining, Incubation, Recombinant, Concentration Assay, Activity Assay, Fluorescence

Journal: eLife

Article Title: Clearance of senescent decidual cells by uterine natural killer cells in cycling human endometrium

doi: 10.7554/eLife.31274

Figure Lengend Snippet: ( A ) Pearson’s correlation analysis of SAβG activity in 75 matched undifferentiated primary cultures and cultures decidualized for 8 days. ( B ) Representative SAβG staining in undifferentiated (Day 0) and decidualizing EnSCs (Day 8) following 4 days of pretreatment with vehicle, dasatinib (250 nM) or palbociclib (1 μM). Scale bar = 100 µm. ( C ) PRL and IGFBP1 mRNA expression in response to pretreatment with vehicle, dasatinib or palbociclib. The cultures then remained undifferentiated or were decidualized for 8 days. ( D ) IL-8, IL-6 and GROα secretion was measured every 48 hr in the supernatant of primary EnSCs decidualized for the indicated time-points following pretreatment with vehicle, dasatinib or palbociclib. ( E ) Colony forming unit (CFU) activity in paired EnSC cultures that either remain undifferentiated (Day 0) or were decidualized for 8 days (n = 10). ( F ) Left panel: representative clonogenic assays established from EnSC cultures first pretreated with vehicle, dasatinib or palbociclib and then decidualized for 8 days. Right panel: CFU activity in EnSC cultures first pretreated with vehicle, dasatinib or palbociclib and then decidualized for 8 days. Data are mean ±SEM of 3 biological replicates unless stated otherwise. *p < 0.05, **p < 0.01 and ***p < 0.001. Different letters above the error bars indicate that those groups are significantly different from each other at p<0.05. 10.7554/eLife.31274.012 Figure 4—source data 1. Functions of senescent decidual cells.

Article Snippet: ELISAs were performed exactly as per manufacturer’s instructions (DuoSet ELISA kits for IL-8 (D8000C), IL-15 (D1500), GROα (DY275) and IL-6 (DY206); Bio-Techne, Abingdon, UK).

Techniques: Activity Assay, Staining, Expressing

Journal: Aging cell

Article Title: Antagonizing peroxisome proliferator-activated receptor γ facilitates M1-to-M2 shift of microglia by enhancing autophagy via the LKB1-AMPK signaling pathway.

doi: 10.1111/acel.12774

Figure Lengend Snippet: FIGURE 1 Antagonizing PPARc prevents LPS-induced M1 microglial activation and facilitates microglial polarization to the M2 phenotype. Microglia were pretreated 15 min with LPS (0.01 lg/ml) and then incubated with PPARc antagonist T0070907 (0.1 lM) for 24 hr. (a) The PPARc antagonist T0070907 suppressed the amoeboid “activated” morphology of microglia induced by LPS. The PPARc antagonist T0070907 reversed LPS-induced mRNA expressions of M1 markers (CD86, Cox-2, iNOS, IFN-c, IL-1b, TNF-a, and CCL2) (b–i) and mRNA expressions of M2 markers (CD206, IL-4, G-CSF, GM-CSF, IGF-1, TGF-b1, TGF-b2, and TGF-b3) in microglial cells (j–q). The ratios of p-NFjB to NFjB (r, s) and p-IKKb to IKKb (t, u) in microglial cells were determined by Western blotting. Data are presented as mean SEM, n ≥4, *p < .05, **p < .01, ***p < .001, compared to the Con group; #p < .05, ##p < .01, ###p < .001, compared to the LPS group

Article Snippet: Primary microglial cells were respectively treated with the PPARc antagonist rosiglitazone (Selleck Chemicals, Houston, TX, USA) or the PPARc antagonist T0070907 (Selleck Chemicals) diluted in dimethylsulfoxide (DMSO; Sigma) with a final concentration of 0.1 lM, which was applied after the treatment with LPS (0.01 lg/ml; Sigma-Aldrich, St. Louis, MO, USA).

Techniques: Activation Assay, Incubation, Western Blot

Journal: Aging cell

Article Title: Antagonizing peroxisome proliferator-activated receptor γ facilitates M1-to-M2 shift of microglia by enhancing autophagy via the LKB1-AMPK signaling pathway.

doi: 10.1111/acel.12774

Figure Lengend Snippet: FIGURE 2 The PPARc antagonist T0070907 improves autophagy in microglia. (a–d) The PPARc antagonist T0070907 prevented LPS- induced decreases in LC3-II/LC3-I, Beclin1, and Atg5 and LPS-induced increases in P62. Data are presented as mean SEM, n ≥4, *p < .05, **p < .01, compared to the Con group; #p < .05, ##p < .01, compared to the LPS group. (e) Representative confocal images of LC3 puncta formation; LC3 (green) was colocalized with Iba1 (red) in cells treated with the PPARc antagonist T0070907 and LPS. Nuclei counterstained with Hoechst 33342. Scale bar = 20 lm. (f) Representative TEM images. Scale bar = 2 lm. High magnification of the boxed areas is shown below. Scale bar = 500 nm. Autophagosomes (blue arrows), autolysosomes (red arrows), and multilamellar body (yellow arrow). (g) Confocal microscopy analysis was used to measure autophagosomes and autolysosomes by monitoring the distribution and alteration of mCherry and EGFP fluorescent signals from mCherry-EGFP-LC3B. Scale bar = 20 lm. Microglia were pretreated with 3-MA (500 lM) or vehicle for 15 min, followed by treatment with 0.01 lg/ml LPS and 0.1 lM PPARc antagonist T0070907 for 24 hr. (h) Immunofluorescence staining of LC3 (green), CD63 (red), and nuclei (blue) in LPS, LPS+T0070907, and LPS+T0070907 + 3-MA groups. Scale bar = 20 lm. The protein expressions of LC3 (i), Beclin1 (j), p62 (k), and Atg (l) were determined by Western blotting. Data are presented as mean SEM, n ≥4, *p < .05, **p < .01, ***p < .001, compared to the Con group; #p < .05, ##p < .01, ###p < .001, compared to the LPS group; †p < .05, ††p < .01, †††p < .001, compared to the LPS+T0070907 group

Article Snippet: Primary microglial cells were respectively treated with the PPARc antagonist rosiglitazone (Selleck Chemicals, Houston, TX, USA) or the PPARc antagonist T0070907 (Selleck Chemicals) diluted in dimethylsulfoxide (DMSO; Sigma) with a final concentration of 0.1 lM, which was applied after the treatment with LPS (0.01 lg/ml; Sigma-Aldrich, St. Louis, MO, USA).

Techniques: Confocal Microscopy, Immunofluorescence, Staining, Western Blot

Journal: Aging cell

Article Title: Antagonizing peroxisome proliferator-activated receptor γ facilitates M1-to-M2 shift of microglia by enhancing autophagy via the LKB1-AMPK signaling pathway.

doi: 10.1111/acel.12774

Figure Lengend Snippet: FIGURE 3 Antagonizing PPARc reverses LPS-mediated inhibition of autophagy in microglial cells by activating AMPK. Microglial cells were pretreated with 0.01 lg/ml LPS, followed by treatment with 0.1 lM PPARc antagonist T0070907 for 24 hr. The upstream regulatory protein levels of autophagy, AMPK (a, b), ULK1 (a, c), and mTOR (a, d) were analyzed by Western blotting. Data are presented as mean SEM, n ≥4, *p < .05, **p < .01, ***p < .001, compared to the Con group; #p < .05, ##p < .01, ###p < .001, compared to the LPS group; †p < .05, ††p < .01, †††p < .001, compared to the LPS+T0070907 group. (e–h) The PPARc antagonist T0070907 increased the phosphorylation of LKB1, but it did not change the phosphorylation of CaMKKb and TAK1 in the microglial cells treated with LPS. (i) An anti-LKB1 antibody was used for Dynabeads Protein G immunoprecipitation, and it detected the immunoprecipitates of MO25 and STRAD by Western blotting. (j) An anti-MO25 antibody was used for Dynabeads Protein G immunoprecipitation, and it detected the immunoprecipitates of LKB1 and STRAD by Western blotting

Article Snippet: Primary microglial cells were respectively treated with the PPARc antagonist rosiglitazone (Selleck Chemicals, Houston, TX, USA) or the PPARc antagonist T0070907 (Selleck Chemicals) diluted in dimethylsulfoxide (DMSO; Sigma) with a final concentration of 0.1 lM, which was applied after the treatment with LPS (0.01 lg/ml; Sigma-Aldrich, St. Louis, MO, USA).

Techniques: Inhibition, Western Blot, Phospho-proteomics, Immunoprecipitation

Journal: Aging cell

Article Title: Antagonizing peroxisome proliferator-activated receptor γ facilitates M1-to-M2 shift of microglia by enhancing autophagy via the LKB1-AMPK signaling pathway.

doi: 10.1111/acel.12774

Figure Lengend Snippet: FIGURE 4 Phosphorylation of LKB1–AMPK is necessary for T0070907-mediated upregulation of autophagy. (a, b) Below the concentration of 0.05 lM, radicicol did not influence the viability of the microglia. After LPS stimulation for 15 min, the LKB1 inhibitor radicicol (0.025 and 0.05 lM) was applied for 15 min before treatment with PPARc antagonist T0070907 for 24 hr. (c–g) Radicicol turned over the protein expressions of LKB1, AMPK, LC3, Beclin1, and p62 in the microglia treated with LPS and T0070907. Data are presented as mean SEM, n ≥4, *p < .05, **p < .01, ***p < .001, compared to the Con group; #p < .05, ##p < .01, ###p < .001, compared to the LPS group; †p < .05, ††p < .01, compared to the LPS+T0070907 group. (h) Immunofluorescence staining with of LC3 (green), CD63 (red), and nuclei (blue) in the LPS, LPS+T0070907, and LPS+T0070907 + radicicol groups. Scale bar = 20 lm. (i) Quantitation of Western blotting data showing declines in LKB1, which was used to observe the efficiency of transfection. Microglia were pretreated with si-LKB1 (500 nM) or vehicle for 24 hr and then stimulated with LPS for 15 min and treated with PPARc antagonist T0070907 for 24 hr. LKB1 siRNA prevented T0070907-induced increases in LC3-II/LC3-I (j), Beclin1 (k), and Atg5 (m) and T0070907-induced decreases in P62 (l). Data are presented as mean SEM, n ≥4, **p < .01, ***p < .001 compared to the LPS group in the Ctrl; ##p < .01, ###p < .001, compared to the LPS+T0070907 group in the Ctrl

Article Snippet: Primary microglial cells were respectively treated with the PPARc antagonist rosiglitazone (Selleck Chemicals, Houston, TX, USA) or the PPARc antagonist T0070907 (Selleck Chemicals) diluted in dimethylsulfoxide (DMSO; Sigma) with a final concentration of 0.1 lM, which was applied after the treatment with LPS (0.01 lg/ml; Sigma-Aldrich, St. Louis, MO, USA).

Techniques: Phospho-proteomics, Concentration Assay, Immunofluorescence, Staining, Quantitation Assay, Western Blot, Transfection

Journal: Aging cell

Article Title: Antagonizing peroxisome proliferator-activated receptor γ facilitates M1-to-M2 shift of microglia by enhancing autophagy via the LKB1-AMPK signaling pathway.

doi: 10.1111/acel.12774

Figure Lengend Snippet: FIGURE 5 Blocking LKB1 reverses the T0070907-mediated transition between M1 and M2 phenotypes by suppressing NFjB. After LPS stimulation for 15 min, the LKB1 inhibitor radicicol (0.025 and 0.05 lM) was applied 15 min before treatment with PPARc antagonist T0070907 for 24 hr. (a) Fluorescence-activated cell sorting analysis of the microglia in the LPS, LPS+T0070907, and LPS+T0070907 + radicicol groups. Surface expression of CD86 and CD206 was detected in microglia by flow cytometry. The percentage of CD86 (b) and CD206 (c) cells in the microglia was determined. pHrodo™Green Zymosan BioParticles were added to the cells and imaged after 30, 60, 90, 120, and 150 min. The green staining in the microglial cells was due to Cell Tracker™Green. (d) The microglial cells showed the time course of red fluorescence increased, documenting the accumulation of pHrodo-conjugated zymosan bioparticles (1 lm in diameter) in the intracellular acidic environment corresponding to phagosomes. (e) The proportion between the red-stained cells and the total cells was calculated. Data are presented as mean SEM, n = 3, ###p < .001 compared to the LPS group in each time point; †p < .05, †††p < .001, compared to the LPS+T0070907 group in each time point. Nonfluorescence appeared at a neutral pH outside of the cell. Scale bar = 20 lm. Radicicol (f) and knocking down LKB1 (i) reversed the PPARc antagonist T0070907-induced changes in the protein expression of the M1 markers (iNOS, green) and the M2 markers (CD206, red) by immunofluorescence staining. Scale bar = 20 lm. (g, h) Western blotting showed that radicicol prevented the decreases in iNOS and the increases in CD206 induced by T0070907. Data are presented as mean SEM, n = 4, ***p < .001, compared to the Con group; ##p < .01, ###p < .001, compared to the LPS group; †p < .05, †††p < .001, compared to the LPS+T0070907 group. (j and k) Knocking down LKB reversed the PPARc antagonist T0070907-induced changes in the protein expressions of iNOS and CD206 by Western blotting. Data are presented as mean SEM, n = 4, ***p < .001, compared to Con group, respectively in Ctrl or in si-LKB1; ###p < .001, compared to LPS group, respectively in Ctrl or in si-LKB1

Article Snippet: Primary microglial cells were respectively treated with the PPARc antagonist rosiglitazone (Selleck Chemicals, Houston, TX, USA) or the PPARc antagonist T0070907 (Selleck Chemicals) diluted in dimethylsulfoxide (DMSO; Sigma) with a final concentration of 0.1 lM, which was applied after the treatment with LPS (0.01 lg/ml; Sigma-Aldrich, St. Louis, MO, USA).

Techniques: Blocking Assay, Fluorescence, FACS, Expressing, Flow Cytometry, Staining, Immunofluorescence, Western Blot

Journal: Aging cell

Article Title: Antagonizing peroxisome proliferator-activated receptor γ facilitates M1-to-M2 shift of microglia by enhancing autophagy via the LKB1-AMPK signaling pathway.

doi: 10.1111/acel.12774

Figure Lengend Snippet: FIGURE 6 Knocking down PPARc reduces the inflammatory response by promoting LKB–AMPK activation. (a) Labeled (FM) with green were used to observe the efficiency of transfection reagent in BV2 cells. Scale bar = 1,000 lm. (b) Images of dissociated microglial cells exposed to scrambled (Con, top panels) or PPARc siRNA, and labeled with antibodies to Iba1 (red) and PPARc (green), and colabeled with a nuclear stain (blue). Scale bar = 100 lm. (c) Quantitation of Western blotting data showing declines in PPARc. Statistical analysis was performed using one-way ANOVA followed by Bonferroni’s post hoc test. Data are presented as mean SEM, n = 5, $$p < .01 compared to the NC group. Microglia were pretreated with si-PPARc (500 nM) or vehicle for 24 hr and then stimulated with LPS for 15 min and treated with PPARc antagonist T0070907 for 24 hr. (d) PPARc antagonist T0070907 did not influence PPARc siRNA-mediated protein expressions of the M1 marker (iNOS, green) and the M2 marker (CD206, red). Scale bar = 20 lm. The experiment was repeated three times. (e, f) Western blotting was used to quantify the expressions of iNOS and CD206 in each group. Data are presented as mean SEM, n = 4, *p < .05, ***p < .001, compared to the Con group, respectively in Ctrl or in si-PPARc; ###p < .001, compared to the LPS group, respectively in Ctrl in Ctrl or in si-PPARc. PPARc siRNA inhibited the activation of NFjB and IKKb (e, f, and g). PPARc siRNA was also associated with a rise in p- LKB1 (e, h) and p-AMPK (e, i). The PPARc antagonist T0070907 did not affect the efficacy of PPARc siRNA. Data are presented as mean SEM, n ≥5, **p < .01, ***p < .001, compared to the LPS group in the control

Article Snippet: Primary microglial cells were respectively treated with the PPARc antagonist rosiglitazone (Selleck Chemicals, Houston, TX, USA) or the PPARc antagonist T0070907 (Selleck Chemicals) diluted in dimethylsulfoxide (DMSO; Sigma) with a final concentration of 0.1 lM, which was applied after the treatment with LPS (0.01 lg/ml; Sigma-Aldrich, St. Louis, MO, USA).

Techniques: Activation Assay, Labeling, Transfection, Staining, Quantitation Assay, Western Blot, Marker, Control

Journal: bioRxiv

Article Title: Therapeutic targeting of SETD2-deficient cancer cells with the small-molecule compound RITA

doi: 10.1101/2021.07.08.451582

Figure Lengend Snippet: RITA induces replication stress in SETD2-deficient cancer cells. (A) Confocal microscopy of phospho-RPA Ser33 (red) and 53BP1 (green) foci in U2OS cells after RITA treatment. Representative images from 3 independent experiments are shown. (B) Quantification of the average number of foci per nucleus from (A) . At least 70 nuclei per biological replicate (n = 3) were counted for statistical analysis. Data shown as mean ± SD. P-values were calculated using the two-tailed Student’s t-test. *p < 0.05, **p < 0.01. RITA induces replication stress in SETD2-deficient cancer cells. (C) DNA fibre assay of parental and SETD2-CRISPR U2OS. Cells were treated as indicated. Representative images from 2 independent experiments are shown. (D) Quantification of replication fork velocity from (C) . At least 200 fibres per biological replicate (n = 3) were measured for statistical analysis. Data shown as median + interquartile range. P-values were calculated using the two-tailed Student’s t-test. N.S. = non-significant, ****p < 0.000001.

Article Snippet: The primary antibodies used for immunofluorescence are: phospho-RPA (Ser33) (Bethyl Laboratories), 53BP1 (GeneTex), and γH2AX (Novus).

Techniques: Confocal Microscopy, Two Tailed Test, CRISPR

Journal: bioRxiv

Article Title: Therapeutic targeting of SETD2-deficient cancer cells with the small-molecule compound RITA

doi: 10.1101/2021.07.08.451582

Figure Lengend Snippet: Loss or inhibition of the phenol sulphotransferase SULT1A1 abolishes RITA-induced phenotypes. (A) Dose response viability curves for SETD2-CRISPR U2OS cells treated with RITA after the addition of non-targeting control (siNT) or SULT1A1 siRNA (siSULT1A1). Data are shown as mean ± SD. (B) Western blot of SULT1A1 in U2OS cells in the presence of non-targeting control (siNT) or SULT1A1 siRNA (siSULT). (C) Dose response viability curves for U2OS cells treated with RITA in the presence or absence of DCNP. Data are shown as mean ± SD. (D) Clonogenic survival assay for U2OS cells in the presence of the indicated compounds. Data are shown as mean ± SD. P-values were calculated using the two-tailed Student’s t-test. **p < 0.01, ****p < 0.0001. (E) Western blot of total p53, phospho-p53 (Ser15), and γH2AX in U2OS cells after the indicated treatments. (F) Western blot of procaspase 3 and cleaved caspase 3 in U2OS cells after the indicated treatments. (G) Cell cycle profiles of U2OS cells in the presence of the indicated compounds. Data shown as mean ± SD. (H) Confocal microscopy of phospho-RPA Ser33 (red) and 53BP1 (green) foci in U2OS cells after the indicated treatments. Representative images from 3 independent experiments are shown. (I) Quantification of the average number of foci per nucleus from (I) . At least 80 nuclei per biological replicate (n = 3) were counted for statistical analysis. Data shown as mean ± SD. P-values were calculated using the two-tailed Student’s t-test. *p < 0.05.

Article Snippet: The primary antibodies used for immunofluorescence are: phospho-RPA (Ser33) (Bethyl Laboratories), 53BP1 (GeneTex), and γH2AX (Novus).

Techniques: Inhibition, CRISPR, Control, Western Blot, Clonogenic Cell Survival Assay, Two Tailed Test, Confocal Microscopy

Journal: The Journal of cell biology

Article Title: ARFRP1 functions upstream of ARL1 and ARL5 to coordinate recruitment of distinct tethering factors to the trans-Golgi network.

doi: 10.1083/jcb.201905097

Figure Lengend Snippet: Figure 2. Both ARL5 and ARFRP1 are required for localization of GARP to the TGN. (A) KO of TGN-localized small GTPases in HeLa cells confirmed by immunoblot analysis with antibodies to the proteins indicated on the right. In this figure and subsequent figures, ARL5 KO represents KO of both ARL5A and ARL5B, and RAB6 KO represents KO of both RAB6A and RAB6B. α-Tubulin was used as a loading control. The positions of molecular mass markers are indicated on the left. (B) Immunofluorescence microscopy of WT and KO HeLa cells transfected with a plasmid encoding VPS54-13Myc and stained for the Myc epitope (red), giantin (green), and nuclei (DAPI; blue). Scale bars: 10 μm. Insets are magnified views of the boxed areas. Inset scale bars: 5 μm. (C) Quantification of the percentage of cells exhibiting VPS54-13Myc staining at the TGN. Values are the mean ± SEM from three independent experiments. More than 100 cells per sample were counted in each experiment. The statistical significance of the differences relative to WT cells was determined using Dunnett’s test. **, P < 0.01; ***, P < 0.001.

Article Snippet: The following antibodies were used for immunoblotting and/or immunofluorescence microscopy: rabbit anti-VPS51 (HPA039650; Atlas Antibodies), rabbit anti-VPS52 made in our laboratory (Pérez-Victoria et al., 2008), rabbit anti-VPS53 (HPA024446; Atlas Antibodies), mouse anti-VPS50 (FLJ20097, monoclonal antibody M01, 2D11; Abnova), mouse anti-Myc epitope (9E10; Santa Cruz Biotechnology), sheep anti-TGN46 (AHP500G; Bio-Rad), mouse anti–β-actin (G043; Applied Biological Materials), rabbit antigiantin (ab80864; Abcam), mouse HRP-conjugated anti-α-tubulin (DM1A; Santa Cruz Biotechnology), rabbit anti-ARL1 (16012-1-AP; Proteintech), rabbit anti-ARFRP1 (PA5-50606; Thermo Fisher Scientific), mouse anti-ARL5A (sc-514680; Santa Cruz Biotechnology), rabbit anti-RAB6A (GTX110646; GeneTex), rabbit anti-GCC88 (HPA021323; Sigma-Aldrich), rabbit anti-GCC185 (HPA035849; Sigma-Aldrich), mouse anti-Golgin-245 (611281; BD Biosciences), mouse anti-GM130 (610822; BD Biosciences), mouse anti–Golgin-97 (A-21270; Thermo Fisher Scientific), rabbit anti-TMF1 (HPA008729, Sigma-Aldrich), monoclonal HRP-conjugated anti-GFP (Miltenyi Biotec Inc.), rabbit anti-GFP (A-11122; Thermo Fisher Scientific), HRP-conjugated goat anti-rabbit and donkey anti-mouse antibodies (Jackson ImmunoResearch), HRP-conjugated donkey antisheep (R&D Systems), and Alexa Fluor–conjugated secondary antibodies for immunostaining (Thermo Fisher Scientific).

Techniques: Western Blot, Control, Immunofluorescence, Microscopy, Transfection, Plasmid Preparation, Staining

Journal: The Journal of cell biology

Article Title: ARFRP1 functions upstream of ARL1 and ARL5 to coordinate recruitment of distinct tethering factors to the trans-Golgi network.

doi: 10.1083/jcb.201905097

Figure Lengend Snippet: Figure 5. Small GTPases required for localization of golgins to the TGN. (A) Immunofluorescence microscopy of WT, ARL1-KO, ARFRP1-KO, ARL5-KO, and RAB6-KO cells immunostained for endogenous Golgin-245, Golgin-97, GCC88, GCC185, or TMF1 and counterstained with DAPI (blue). Scale bars: 10 μm. Insets are magnified views of the boxed areas. Inset scale bars: 5 μm. Notice that ARL1 KO or ARFRP1 KO caused complete disappearance of Golgin-245 and GCC88, and a partial decrease in the intensity of Golgin-97, at the TGN; quantification in 10 cells per sample in three independent experiment showed that Golgin-97 decrease was 75.6% ± 2.4% in ARL1-KO cells and 55.0% ± 1.9% in ARFRP1-KO cells. (B) SDS-PAGE and immunoblot analysis of endogenous golgins and α-tubulin (loading control) in WT and KO cells. The positions of molecular mass markers are indicated on the left. (C) Immunofluorescence microscopy of RAB6- KO cells transfected with a plasmid encoding GFP-tagged mouse Rab6A (green), immunostained for endogenous GCC185 and TMF1 (red), and counterstained with DAPI (blue). Cells were examined for GFP fluorescence by confocal microscopy. Scale bars: 10 μm.

Article Snippet: The following antibodies were used for immunoblotting and/or immunofluorescence microscopy: rabbit anti-VPS51 (HPA039650; Atlas Antibodies), rabbit anti-VPS52 made in our laboratory (Pérez-Victoria et al., 2008), rabbit anti-VPS53 (HPA024446; Atlas Antibodies), mouse anti-VPS50 (FLJ20097, monoclonal antibody M01, 2D11; Abnova), mouse anti-Myc epitope (9E10; Santa Cruz Biotechnology), sheep anti-TGN46 (AHP500G; Bio-Rad), mouse anti–β-actin (G043; Applied Biological Materials), rabbit antigiantin (ab80864; Abcam), mouse HRP-conjugated anti-α-tubulin (DM1A; Santa Cruz Biotechnology), rabbit anti-ARL1 (16012-1-AP; Proteintech), rabbit anti-ARFRP1 (PA5-50606; Thermo Fisher Scientific), mouse anti-ARL5A (sc-514680; Santa Cruz Biotechnology), rabbit anti-RAB6A (GTX110646; GeneTex), rabbit anti-GCC88 (HPA021323; Sigma-Aldrich), rabbit anti-GCC185 (HPA035849; Sigma-Aldrich), mouse anti-Golgin-245 (611281; BD Biosciences), mouse anti-GM130 (610822; BD Biosciences), mouse anti–Golgin-97 (A-21270; Thermo Fisher Scientific), rabbit anti-TMF1 (HPA008729, Sigma-Aldrich), monoclonal HRP-conjugated anti-GFP (Miltenyi Biotec Inc.), rabbit anti-GFP (A-11122; Thermo Fisher Scientific), HRP-conjugated goat anti-rabbit and donkey anti-mouse antibodies (Jackson ImmunoResearch), HRP-conjugated donkey antisheep (R&D Systems), and Alexa Fluor–conjugated secondary antibodies for immunostaining (Thermo Fisher Scientific).

Techniques: Immunofluorescence, Microscopy, SDS Page, Western Blot, Control, Transfection, Plasmid Preparation, Fluorescence, Confocal Microscopy

Journal: Cell Death Discovery

Article Title: Transcription factor 7-like 1 dysregulates keratinocyte differentiation through upregulating lipocalin 2

doi: 10.1038/cddiscovery.2016.28

Figure Lengend Snippet: The effect of Tcf7l1 depletion on LCN2 expression in HFKs. HFKs were stimulated with calcium chloride (1.2 mM, 7 days) after siRNA transfection. ( a and b ) Intracellular ( a ) and soluble ( b ) LCN2 levels were evaluated by western blot and ELISA, respectively. ( c ) By immunofluorescence, the expression of involucrin and loricrin were detected in HFKs that were transfected with Tcf7l1 or control siRNA. ( d and e ) The expression of involucrin and loricrin was also detected in HFKs by western blot. Data show mean±S.E.M. of triplicate cultures ( n =3). Statistical differences were tested with one-way ANOVA followed by a two-tailed t -test. Representative images are shown. Scale bar=10 μ m. * P <0.05; ** P <0.01.

Article Snippet: For confocal analysis, rabbit anti-involucrin (Alexa Fluor 555-conjugated; Bioss), anti-loricrin (Cy5-conjugated) (Biorbyt LLC, San Francisco, CA, USA) or anti-cytokeratin 1 (Alexa Fluor 488-conjugated) (Abcam, Cambridge, MA, USA) antibodies were used by following the standard method.

Techniques: Expressing, Transfection, Western Blot, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Two Tailed Test

Journal: Cell Death Discovery

Article Title: Transcription factor 7-like 1 dysregulates keratinocyte differentiation through upregulating lipocalin 2

doi: 10.1038/cddiscovery.2016.28

Figure Lengend Snippet: The effect of Tcf7l1 overexpression on HFKs. After the transfection of Tcf7l1-inserted pcDNA vector, HFKs were stimulated with calcium chloride (1.2 mM, 7 days). ( a and b ) By qRT-PCR ( a ) and western blot ( b ), HFKs expressed more lipocalin 2 (LCN2) upon Tcf7l1 pcDNA transfection. ( c and d ) Both cell apoptosis ( c ) and proliferation ( d ) were determined in HFKs that were either transfected with Tcf7l1 pcDNA or not. Cell apoptosis and proliferation were also visualized by TUNEL and fluorescent Ki-67 staining, respectively. ( e ) qRT-PCR was performed for the mRNA levels of involucrin, loricrin and caspase-14 in HFKs. Data show mean±S.E.M. of triplicate cultures ( n =3). Statistical differences were tested with one-way ANOVA followed by a two-tailed t -test. Scale bar=10 μ m. In ( e ), * P <0.05, ** P <0.01, compared with nontreated groups accordingly; Δ P <0.05, compared with calcium alone-treated HFKs accordingly.

Article Snippet: For confocal analysis, rabbit anti-involucrin (Alexa Fluor 555-conjugated; Bioss), anti-loricrin (Cy5-conjugated) (Biorbyt LLC, San Francisco, CA, USA) or anti-cytokeratin 1 (Alexa Fluor 488-conjugated) (Abcam, Cambridge, MA, USA) antibodies were used by following the standard method.

Techniques: Over Expression, Transfection, Plasmid Preparation, Quantitative RT-PCR, Western Blot, TUNEL Assay, Staining, Two Tailed Test

Journal: Cell Death Discovery

Article Title: Transcription factor 7-like 1 dysregulates keratinocyte differentiation through upregulating lipocalin 2

doi: 10.1038/cddiscovery.2016.28

Figure Lengend Snippet: The effect of LCN2 inhibition on HFKs. ( a and b ) By western blot, involucrin and loricrin expression was determined in Tcf7l1-transfected HFKs that were then treated with LCN2 or control siRNA. ( c and d ) Similarly, involucrin and loricrin were assessed in E6/E7-transfected HFKs that were treated with neutralizing anti-LCN2 antibody or isotype control (2 μ g/ml, 2 days). ( e and f ) By immunofluorescence ( e ) and western blot ( f ), keratin 1 was detected in HFKs that were transfected with Tcf7l1 vector and then LCN2 or control siRNA. ( g ) HFKs were pre-transfected with Tcf7l1 vector, and then transfected with LCN2 siRNA. Cell apoptosis was quantitated by the CaspaTag caspase 3, 7 method. Data show mean±S.E.M. of triplicate cultures ( n =3). Statistical differences were tested with one-way ANOVA followed by a two-tailed t -test. Representative images are shown. Scale bar=10 μ m. * P <0.05; ** P <0.01.

Article Snippet: For confocal analysis, rabbit anti-involucrin (Alexa Fluor 555-conjugated; Bioss), anti-loricrin (Cy5-conjugated) (Biorbyt LLC, San Francisco, CA, USA) or anti-cytokeratin 1 (Alexa Fluor 488-conjugated) (Abcam, Cambridge, MA, USA) antibodies were used by following the standard method.

Techniques: Inhibition, Western Blot, Expressing, Transfection, Immunofluorescence, Plasmid Preparation, Two Tailed Test

Journal: Biocell

Article Title: Stretch Enhances Secretion of Extracellular Vehicles from Airway Smooth Muscle Cells via Endoplasmic Reticulum Stress Signaling in Relation to Ventilator-Induced Lung Injury

doi: 10.32604/biocell.2025.063869

Figure Lengend Snippet: Figure 2: The effect of stretch on EVs and endoplasmic reticulum (ER) stress in ASMCs. (A) Left to right: representative immunofluorescence images of CD63/HSPA5 in ASMCs acquired with confocal microscopy (×100 objective), the quantified CD63/HSPA5 puncta number inside ASMCs (ImageJ), and Pearson coefficient of the pixel-intensity correlation of CD63/HSPA5 in ASMCs (ImageJ, scale bar = 200 μm); (B) Protein expression of CD63/HSPA5 in ASMCs (WB); (C) Size distribution and quantified counts, as well as protein concentration of EVs in ASMCs (NTA and BCA). ER: endoplasmic reticulum; HSPA5: biomarker protein of ER stress; TUDCA: tauroursodeoxycholic acid, ER stress inhibitor; NTA, nanoparticle tracking analysis; BCA: bicinchoninic acid assay; data present as means ± SD, experiments were repeated three times (n = 3), **p < 0.01 compared with Static, ##p < 0.01 compared with Stretch

Article Snippet: Afterwards it was immersed in 10% horse serum (Sigma, #H0146) in PBS as a blocking solution for 1 h at RT, washed 3 times in PBS for 5 min, and then incubated with HSPA5 antibody (1:100, #BA2042, BOSTER) in PBS containing 5% horse serum for 1 h at RT.

Techniques: Immunofluorescence, Confocal Microscopy, Expressing, Protein Concentration, Biomarker Discovery, Acid Assay

Journal: Biocell

Article Title: Stretch Enhances Secretion of Extracellular Vehicles from Airway Smooth Muscle Cells via Endoplasmic Reticulum Stress Signaling in Relation to Ventilator-Induced Lung Injury

doi: 10.32604/biocell.2025.063869

Figure Lengend Snippet: Figure 6: The effect of mechanical ventilation on secretion of EVs, ER stress, airway inflammation and injury in mouse models of VILI. (A) Representative images of lung tissue slides with hematoxylin and eosin (HE) or immunohisto- chemistry (IHC, HSPA5) staining (scale bar = 100 μm, arrows indicate alveolar collapse, arrowhead indicate HSPA5 expression). Healthy, MV, MV + TUDCA/GW4869 indicates mice breathed spontaneously, under 18 mL/kg mechanical ventilation (MV), MV with pretreatment of TUDCA/GW4869, respectively; (B) Protein expression of HSPA5 in lung tissue with IHC staining from C57BL/6 mice treated with different conditions, respectively; (C–E) Size distribution and quantified count, HSPA5, as well as protein concentration of EVs isolated from bronchoalveolar lavage fluid (BALF) of mice treated with different conditions; (F) Relative TGF-β1 and IL-10 secretion in BALF from mice treated with different conditions. Data present as means ± SD, experiments were repeated six times (n = 6), ** p < 0.01 compared with Healthy, #p < 0.05, ##p < 0.01 compared with MV

Article Snippet: Afterwards it was immersed in 10% horse serum (Sigma, #H0146) in PBS as a blocking solution for 1 h at RT, washed 3 times in PBS for 5 min, and then incubated with HSPA5 antibody (1:100, #BA2042, BOSTER) in PBS containing 5% horse serum for 1 h at RT.

Techniques: Immunohistochemistry, Staining, Expressing, Protein Concentration, Isolation

Journal: Journal of Virology

Article Title: PCV2 Induces Reactive Oxygen Species To Promote Nucleocytoplasmic Translocation of the Viral DNA Binding Protein HMGB1 To Enhance Its Replication

doi: 10.1128/JVI.00238-20

Figure Lengend Snippet: PCV2 infection led to translocation of HMGB1 from nuclei to cytoplasmic compartments. PK-15 cells and porcine monocytic cells (3D4/31) were infected for 36 h with PCV2 (MOI = 1) or mock infected as a control. (A) Confocal imaging of HMGB1 distribution in PCV2-infected cells immunostained with anti-HMGB1 (green) and anti-Cap (red) antibodies. Nuclei were labeled with DAPI (blue). Representative micrographic images are shown. (B) Immunoblotting of PCV2 Cap and HMGB1 in nuclear and cytoplasmic extracts from PCV2- or mock-infected PK-15 cells. Histone H3 and GAPDH were used as internal controls for nuclear and cytoplasmic fractions, respectively. (C) The intensity of protein bands was quantified densitometrically using Gel-Pro Analyzer. Ratios of nuclear or cytoplasmic HMGB1 to Histone H3 or GAPDH were quantified, respectively. (D and E) Quantification of hmgb1 mRNA by qPCR in PK-15 and 3D4/31 cells infected with PCV2 for different times using total RNA extracts from the cells. (F and G) Immunoblotting of HMGB1 and PCV2 Cap in the lysates of PK-15 and 3D4/31 cells infected with PCV2 for different times. β-Actin was used as a loading control. The data in panels A, B, F, and G are representative of three independent experiments. Bar charts in panels C, D, and E show means ± SDs from three independent experiments. ns, not significant; *, P < 0.05; **, P < 0.01.

Article Snippet: Approximately 1 mg of total cellular proteins or nuclear proteins was transferred to a 1.5-ml microcentrifuge tube and incubated with 4 μg of rabbit anti-HMGB1 polyclonal antibody (ChIP grade, ab18256) (Abcam) or normal rabbit IgG (Beyotime) for 2 h at 4°C.

Techniques: Infection, Translocation Assay, Imaging, Labeling, Western Blot

Journal: Journal of Virology

Article Title: PCV2 Induces Reactive Oxygen Species To Promote Nucleocytoplasmic Translocation of the Viral DNA Binding Protein HMGB1 To Enhance Its Replication

doi: 10.1128/JVI.00238-20

Figure Lengend Snippet: Overexpression of HMGB1 inhibited PCV2 replication. PK-15 cells were transfected with recombinant plasmid expressing HMGB1 (pHMGB1) or control plasmid (pFlag) for 24 h and then infected with PCV2 (MOI = 1) for 36 h. (A) Effect of HMGB1 overexpression on PCV2 Cap expression as shown by immunoblotting using protein samples from the whole-cell lysates. β-Actin was used as a loading control. The gel shown is representative of three independent experiments. (B) The ratios of band intensity of HMGB1 or PCV2 Cap to β-actin (as shown in panel A). (C) Effect of HMGB1 overexpression on PCV2 orf2 (encoding Cap) transcription measured by qPCR using total RNA extracted from the whole-cell lysates. (D) PCV2 replication in cells overexpressing HMGB1 as assessed by indirect immunofluorescence. Percentages of PCV2-infected cells were calculated as described in the legend for Fig. 2. Relative percentages of PCV2-infected cells in the HMGB1 overexpressing cells are shown with nontransfected but PCV2-infected cells set at 100%. (E) PCV2 genomic DNA copies in cells overexpressing HMGB1 quantified by qPCR using total DNA extracts from whole-cell lysates. Bar charts in panels B, C, D, and E show means ± SDs from three independent experiments. ns, not significant; **, P < 0.01; ***, P < 0.001.

Article Snippet: Approximately 1 mg of total cellular proteins or nuclear proteins was transferred to a 1.5-ml microcentrifuge tube and incubated with 4 μg of rabbit anti-HMGB1 polyclonal antibody (ChIP grade, ab18256) (Abcam) or normal rabbit IgG (Beyotime) for 2 h at 4°C.

Techniques: Over Expression, Transfection, Recombinant, Plasmid Preparation, Expressing, Infection, Western Blot, Immunofluorescence

Journal: Journal of Virology

Article Title: PCV2 Induces Reactive Oxygen Species To Promote Nucleocytoplasmic Translocation of the Viral DNA Binding Protein HMGB1 To Enhance Its Replication

doi: 10.1128/JVI.00238-20

Figure Lengend Snippet: Downregulation of HMGB1 promoted PCV2 replication. PK-15 cells were transfected with hmgb1-specific RNA interference (RNAi) plasmid (sh-HMGB1) or control RNAi plasmid (sh-NC) for 24 h and then infected with PCV2 (MOI= 1) for 36 h. (A) Effect of hmgb1 knockdown on PCV2 Cap expression (β-actin used as a loading control) as shown by immunoblotting using protein samples from the whole-cell lysates. The gel shown is representative of three independent experiments. (B) The ratios of band intensity of HMGB1 or PCV2 Cap to β-actin (as shown in panel A). (C) Effect of hmgb1 knockdown on PCV2 orf2 (encoding Cap) transcription examined by qPCR using total RNA extracted from the whole-cell lysates. (D) PCV2 replication in hmgb1-silenced cells as assessed by indirect immunofluorescence. Representative fluorescence images are shown (top). Percentage of PCV2-infected cells was calculated by dividing the number of PCV2-infected cells by the total cell number in each group (n = 2 images for each experiment per group) that were counted using ImageJ software. Relative percentages of PCV2-infected cells in the hmgb1-silenced cells are shown with nontransfected but PCV2-infected cells set at 100% (bottom). (E) Effect of hmgb1 silencing on PCV2 genomic DNA copies measured by qPCR using total DNA extracts from whole-cell lysates. Bar charts in panels B, C, D, and E show means ± SDs from three independent experiments. ns, not significant; **, P < 0.01.

Article Snippet: Approximately 1 mg of total cellular proteins or nuclear proteins was transferred to a 1.5-ml microcentrifuge tube and incubated with 4 μg of rabbit anti-HMGB1 polyclonal antibody (ChIP grade, ab18256) (Abcam) or normal rabbit IgG (Beyotime) for 2 h at 4°C.

Techniques: Transfection, Plasmid Preparation, Infection, Expressing, Western Blot, Immunofluorescence, Fluorescence, Software

Journal: Journal of Virology

Article Title: PCV2 Induces Reactive Oxygen Species To Promote Nucleocytoplasmic Translocation of the Viral DNA Binding Protein HMGB1 To Enhance Its Replication

doi: 10.1128/JVI.00238-20

Figure Lengend Snippet: Nuclear HMGB1 repressed PCV2 replication. PK-15 cells were transfected with recombinant plasmid expressing HMGB1 (pHMGB1) or control plasmid (pFlag) for 24 h and then infected with PCV2 (MOI = 1) for 36 h. Nuclear and cytoplasmic extracts were prepared for immunoblotting as described in the legend for Fig. 1 Immunoblotting of HMGB1 and PCV2 Cap in the nuclear (A) and cytoplasmic (B) fractions. Histone H3 and GAPDH were used as internal controls for nuclear and cytoplasmic extracts, respectively. Representative images from three independent experiments are shown. The ratios of band intensities of HMGB1 or PCV2 Cap to those of histone H3 (as shown in panel A) in the nuclear fraction (C) or to GAPDH (as shown in panel B) in the cytoplasmic fraction (D). (E) Effect of HMGB1 overexpression on PCV2 orf2 transcription in the nuclei examined by qPCR using total RNA extracted from the nuclear fractions. Results were normalized to histone H3 mRNA in the same samples. (F) PCV2 genomic DNA replication in the nuclei of HMGB1-overexpressing cells quantified by qPCR using total DNA extracted from nuclear fractions. Bar charts in panels C to F show means ± SDs from three independent experiments. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: Approximately 1 mg of total cellular proteins or nuclear proteins was transferred to a 1.5-ml microcentrifuge tube and incubated with 4 μg of rabbit anti-HMGB1 polyclonal antibody (ChIP grade, ab18256) (Abcam) or normal rabbit IgG (Beyotime) for 2 h at 4°C.

Techniques: Transfection, Recombinant, Plasmid Preparation, Expressing, Infection, Western Blot, Over Expression

Journal: Journal of Virology

Article Title: PCV2 Induces Reactive Oxygen Species To Promote Nucleocytoplasmic Translocation of the Viral DNA Binding Protein HMGB1 To Enhance Its Replication

doi: 10.1128/JVI.00238-20

Figure Lengend Snippet: Ethyl pyruvate inhibited nucleocytoplasmic translocation of HMGB1 in PCV2-infected cells. PK-15 cells were mock infected or infected with PCV2 (MOI = 1) with or without ethyl pyruvate (EP; 7.5 mM) treatment. The cell samples were harvested at 36 hpi. (A) Confocal microscopic images show inhibition of nuclear HMGB1 migration into the cytosol by EP. Cells were immunostained for HMGB1 (green) and PCV2 Cap (red), with nuclei stained with DAPI (blue). Bars, 10 μm. (B) Immunoblotting of HMGB1 and PCV2 Cap in the nuclear and cytoplasmic fractions of PCV2-infected and EP-treated cells. Histone H3 and GAPDH were used as internal controls for nuclear and cytoplasmic extracts, respectively. Representative images from three independent experiments are shown. The ratios of band intensities of HMGB1 or PCV2 Cap to those of histone H3 (as shown in panel B, left) in the nuclear fraction (C) or to GAPDH (as shown in panel B, right) in the cytoplasmic fraction (D). (E) Effect of EP on PCV2 genomic DNA replication by qPCR using DNA extracted from nuclei of PCV2-infected cells treated with 7.5 mM EP. Bar charts in panels C, D, and E show means ± SDs from three independent experiments. ns, not significant; **, P < 0.01.

Article Snippet: Approximately 1 mg of total cellular proteins or nuclear proteins was transferred to a 1.5-ml microcentrifuge tube and incubated with 4 μg of rabbit anti-HMGB1 polyclonal antibody (ChIP grade, ab18256) (Abcam) or normal rabbit IgG (Beyotime) for 2 h at 4°C.

Techniques: Translocation Assay, Infection, Inhibition, Migration, Staining, Western Blot

Journal: Journal of Virology

Article Title: PCV2 Induces Reactive Oxygen Species To Promote Nucleocytoplasmic Translocation of the Viral DNA Binding Protein HMGB1 To Enhance Its Replication

doi: 10.1128/JVI.00238-20

Figure Lengend Snippet: N-Acetylcysteine inhibited PCV2-induced HMGB1 translocation from nuclei to cytosol and repressed PCV2 replication. PK-15 cells were mock infected or infected with PCV2 (MOI = 1) for 12 h and then treated with 10 mM N-acetylcysteine (NAC). The cell samples were harvested at 36 hpi. (A) Confocal imaging of HMGB1 distribution in PCV2-infected and NAC-treated cells after the cells were fixed and immunostained for HMGB1 (green) and Cap (red). Nuclei were stained with DAPI (blue). Bars, 10 μm. (B) Blotting of HMGB1 and PCV2 Cap in the nuclear and cytoplasmic extracts of PCV2-infected cells with or without NAC treatment. Histone H3 and GAPDH were used as internal controls for the nuclear and cytoplasmic fractions, respectively. The figure is representative of three independent experiments. The ratios of band intensities of HMGB1 or PCV2 Cap to histone H3 (as shown in panel B, left) in the nuclear fraction (C) or to GAPDH (as shown in panel B, right) in the cytoplasmic fraction (D). (E) Effect of NAC on PCV2 genomic DNA replication by qPCR using DNA extracted from lysates of PCV2-infected cells treated with NAC. (F) Cytosolic ROS levels in PCV2-infected cells with or without treatment by NAC or ethyl pyruvate (EP) as measured by flow cytometry after probing with DCFH-DA. Bar charts in panels C, D, E, and F show means ± SDs from three independent experiments. *, P < 0.05; **, P < 0.01.

Article Snippet: Approximately 1 mg of total cellular proteins or nuclear proteins was transferred to a 1.5-ml microcentrifuge tube and incubated with 4 μg of rabbit anti-HMGB1 polyclonal antibody (ChIP grade, ab18256) (Abcam) or normal rabbit IgG (Beyotime) for 2 h at 4°C.

Techniques: Translocation Assay, Infection, Imaging, Staining, Flow Cytometry

Journal: Journal of Virology

Article Title: PCV2 Induces Reactive Oxygen Species To Promote Nucleocytoplasmic Translocation of the Viral DNA Binding Protein HMGB1 To Enhance Its Replication

doi: 10.1128/JVI.00238-20

Figure Lengend Snippet: Ethyl pyruvate was inhibitory to PCV2 infection. PK-15 cells were mock infected or infected with PCV2 (MOI = 1) with or without ethyl pyruvate (EP; 7.5 mM) treatment. The cell samples were harvested at 36 hpi. (A) Effect of EP on PCV2 replication in PK-15 cells by immunofluorescence. Percentages of PCV2-infected cells were calculated as described in the legend for Fig. 2. Relative percentages of PCV2-infected cells in the EP-treated cells are shown with untreated but PCV2-infected cells set at 100%. (B) Immunoblotting of HMGB1 and PCV2 Cap in whole-cell lysates with β-actin used as a loading control. (C) The ratios of band intensities of HMGB1 or PCV2 Cap to β-actin (as shown in panel B). Bar charts in panels A and C show means ± SDs from three independent experiments. ns, not significant; **, P < 0.01.

Article Snippet: Approximately 1 mg of total cellular proteins or nuclear proteins was transferred to a 1.5-ml microcentrifuge tube and incubated with 4 μg of rabbit anti-HMGB1 polyclonal antibody (ChIP grade, ab18256) (Abcam) or normal rabbit IgG (Beyotime) for 2 h at 4°C.

Techniques: Infection, Immunofluorescence, Western Blot

Journal: Journal of Virology

Article Title: PCV2 Induces Reactive Oxygen Species To Promote Nucleocytoplasmic Translocation of the Viral DNA Binding Protein HMGB1 To Enhance Its Replication

doi: 10.1128/JVI.00238-20

Figure Lengend Snippet: The B box domain of HMGB1 was involved in inhibition of PCV2 replication. (A) Schematic illustration of the full-length and truncated forms of porcine HMGB1 according to its human homolog. All truncated versions, A box, AB box, and B box plus C terminus (B boxCT), were flag tagged. The numbers indicate positions of amino acids. Arrows with C followed by numbers represent key cysteine residues. NLS, nuclear localization signal. PK-15 cells were transfected with recombinant plasmids expressing flag-tagged or full-length HMGB1 for 24 h and then infected with PCV2 (MOI = 1) for 36 h. (B) Numbers of PCV2-infected cells examined by immunofluorescence using anti-Cap monoclonal antibody as the probe (top). Expression of PCV2 Cap and different forms of HMGB1 as assessed by immunoblotting using the whole-cell lysates harvested at 36 hpi and antibodies against Flag, Cap, and HMGB1 (bottom). β-Actin was used as a loading control. The panel B images are representative of three individual experiments. (C) The ratios of band intensities of PCV2 Cap to those of β-actin (as shown at the bottom panel of B). (D) Effect of different HMGB1 truncations on PCV2 DNA replication estimated by qPCR using total DNA extracted from the whole-cell lysate. Bar charts in panels C and D show means ± SDs from three independent experiments. ns, not significant; **, P < 0.01.

Article Snippet: Approximately 1 mg of total cellular proteins or nuclear proteins was transferred to a 1.5-ml microcentrifuge tube and incubated with 4 μg of rabbit anti-HMGB1 polyclonal antibody (ChIP grade, ab18256) (Abcam) or normal rabbit IgG (Beyotime) for 2 h at 4°C.

Techniques: Inhibition, Transfection, Recombinant, Expressing, Infection, Immunofluorescence, Western Blot

Journal: Journal of Virology

Article Title: PCV2 Induces Reactive Oxygen Species To Promote Nucleocytoplasmic Translocation of the Viral DNA Binding Protein HMGB1 To Enhance Its Replication

doi: 10.1128/JVI.00238-20

Figure Lengend Snippet: HMGB1 bound to the Ori region of the PCV2 genome. (A) Binding of porcine HMGB1 to PCV2 DNA using the gel shift assay. PCV2 DNA (500 ng) and various concentrations (0 to 5 μg) of purified His-tagged recombinant HMGB1 were mixed in binding buffer. The DNA-protein mixtures were subjected to 0.8% agarose gel electrophoresis to visualize changes of the DNA motility. (B) Binding of HMGB1 to a specific region of PCV2 DNA: full-length and different fragments of PCV2 genome (orf1, orf2, and Ori) were incubated with recombinant HMGB1 protein to identify the region of PCV2 genome involved in HMGB1 binding. (C) To confirm the Ori region is required for HMGB1 binding, the Ori fragment was combined with orf1 or orf2 (Ori-orf1 or Ori-orf2) that were then compared with orf1 or orf2 alone by the gel shift assay. (D) Immunoprecipitation of purified HMGB1 protein (500 μg) and PCV2 DNA (500 ng) mixture by anti-HMGB1 antibody (rabbit IgG as control) and protein A/G agarose. The precipitates were probed with anti-His and anti-HMGB1 antibodies by immunoblotting. (E) Quantification of PCV2 genomic Ori copies by qPCR in DNA extracts from PCV2 DNA-HMGB1 precipitates (shown in panel D) after DNase pretreatment. (F) Blotting of HMGB1 in immunoprecipitates of whole-cell lysates (WCL) of the PK-15 cells infected with PCV2 (36 h) by anti-HMGB1 (rabbit IgG as control) and protein A/G agarose. (G) Quantification of PCV2 genomic Ori copies by qPCR in DNA extracts from immunoprecipitates of whole-cell lysates (shown in panel F) after DNase pretreatment. (H) Blotting of HMGB1 in immunoprecipitates of nuclear extracts of the PK-15 cells infected with PCV2. (I) Quantification of PCV2 genomic Ori copies in the precipitates shown in panel H. Bar charts in panels E, G, and I show means ± SDs from three independent experiments.

Article Snippet: Approximately 1 mg of total cellular proteins or nuclear proteins was transferred to a 1.5-ml microcentrifuge tube and incubated with 4 μg of rabbit anti-HMGB1 polyclonal antibody (ChIP grade, ab18256) (Abcam) or normal rabbit IgG (Beyotime) for 2 h at 4°C.

Techniques: Binding Assay, Electrophoretic Mobility Shift Assay, Purification, Recombinant, Agarose Gel Electrophoresis, Incubation, Immunoprecipitation, Western Blot, Infection

Journal: Journal of Virology

Article Title: PCV2 Induces Reactive Oxygen Species To Promote Nucleocytoplasmic Translocation of the Viral DNA Binding Protein HMGB1 To Enhance Its Replication

doi: 10.1128/JVI.00238-20

Figure Lengend Snippet: Effect of hydrogen peroxide treatment on subcellular localization of HMGB1 and PCV2 replication. (A) H2O2 treatment promoted nucleocytoplasmic translocation of HMGB1. PK-15 cells were treated with or without N-acetylcysteine (NAC; 10 mM) before adding 50 μM H2O2. Cells were fixed and immunostained with anti-HMGB1 (green) for confocal microscopy. Nuclei were labeled with DAPI (blue). (B) Immunoblotting of HMGB1 in the nuclear and cytoplasmic fractions of PK-15 cells treated with H2O2 and NAC. (C) Confocal imaging of PK-15 cells infected by PCV2 with or without 50 μM H2O2 treatment after immunostaining with anti-HMGB1 (green) and anti-Cap (red) antibodies. (D) Blotting of HMGB1 and PCV2 Cap in the nuclear and cytoplasmic extracts of PCV2-infected cells with or without H2O2 treatment. Histone H3 and GAPDH were used as internal controls for the nuclear and cytoplasmic fractions, respectively. (E) Percentages of PCV2-infected cells were calculated from immunofluorescence images as described in the legend for Fig. 2. Relative percentages of PCV2-infected cells in the H2O2-treated cells are shown with nontreated but PCV2-infected cells set at 100%. Bar chart in panel E shows means ± SDs from three independent experiments. **, P < 0.01.

Article Snippet: Approximately 1 mg of total cellular proteins or nuclear proteins was transferred to a 1.5-ml microcentrifuge tube and incubated with 4 μg of rabbit anti-HMGB1 polyclonal antibody (ChIP grade, ab18256) (Abcam) or normal rabbit IgG (Beyotime) for 2 h at 4°C.

Techniques: Translocation Assay, Confocal Microscopy, Labeling, Western Blot, Imaging, Infection, Immunostaining, Immunofluorescence

Journal: Journal of Virology

Article Title: PCV2 Induces Reactive Oxygen Species To Promote Nucleocytoplasmic Translocation of the Viral DNA Binding Protein HMGB1 To Enhance Its Replication

doi: 10.1128/JVI.00238-20

Figure Lengend Snippet: Schematic illustration of the interaction between PCV2 and HMGB1 in infected cells. HMGB1 in the nucleus restricts PCV2 replication by binding to the Ori region of the PCV2 genome. PCV2 infection causes increased generation of cellular ROS. Increased ROS promotes nucleocytoplasmic translocation of HMGB1 and lessens sequestration of the viral DNA by HMGB1 in the nucleus, thus enhancing PCV2 replication. N-Acetylcysteine (and probably ethyl pyruvate as well) scavenges PCV2-induced ROS and thus increases retention of HMGB1 in the nucleus, leading to sequestration of viral DNA and reduced PCV2 replication.

Article Snippet: Approximately 1 mg of total cellular proteins or nuclear proteins was transferred to a 1.5-ml microcentrifuge tube and incubated with 4 μg of rabbit anti-HMGB1 polyclonal antibody (ChIP grade, ab18256) (Abcam) or normal rabbit IgG (Beyotime) for 2 h at 4°C.

Techniques: Infection, Binding Assay, Translocation Assay

Journal: Science translational medicine

Article Title: Oral delivery of liquid mRNA therapeutics by engineered capsule for treatment of preclinical intestinal disease

doi: 10.1126/scitranslmed.adu1493

Figure Lengend Snippet: ( A ) Enzyme-linked immunosorbent assay (ELISA) analysis of IL-10 concentration in supernatant from Caco-2, MC-38 and Raw 264.7 cells treated with various amounts of PBS, IL-10 -mRNA or IL-10 -mRNA NPs for 24 h. ( B ) Western blot analysis of IL-10 expression in MC-38 cells treated with PBS, IL-10 -mRNA, blank NPs or IL-10 -mRNA NPs for 12 h. IL-10 -mRNA 750 ng/mL. ( C ) Confocal microscopy images of immunofluorescence staining of IL-10 expression in MC-38 cells treated with free IL-10 -mRNA or IL-10 -mRNA NPs. Hoechst (blue) was used to stain the cell nuclei. An Alexa Fluor 647-labeled antibody was used to stain IL-10. ( D ) Schematic illustration of how IL-10 -mRNA NPs directly transfect macrophages, inhibiting the polarization of macrophages to proinflammatory M1 phenotype in the presence of lipopolysaccharide (LPS), an inflammatory stimulator. ( E ) ELISA analysis of anti-inflammatory cytokine IL-10 and proinflammatory cytokine TNF-α and IL-6 in supernatant from RAW 264.7 cells treated with PBS or IL-10 -mRNA NPs (w/wo LPS stimulation) following procedures illustrated in (D). ( F ) Schematic illustration of how IL-10 -mRNA NPs first transfect intestinal epithelial cells, then indirectly induce the repolarization of macrophages from proinflammatory M1 phenotype to anti-proinflammatory M2 phenotype. ( G ) ELISA analysis of IL-10 and TNF-α and IL-6 in supernatant from RAW 264.7 cells treated with supernatant from Caco-2 cells incubated with PBS or IL-10 -mRNA NPs following procedures illustrated in (F). ( H ) Ex vivo images of excised rat gastrointestinal (GI) tract at various time points from 15 min to 6 h post-administration. Rats were orally administered six Cy5-mRNA-RNACaps (L100-55 coated). The left panel shows enlarged images of RNACaps at 1 h and 2 h (images 1–4). Cy5-mRNA: 50 μg per rat. Color scale, 0-255 gray value. ( I ) Confocal microscopy images of intestine sections from rats treated with Cy5-mRNA-RNACaps (purple) for 4 h. Hoechst (blue) was used to stain the cell nuclei. Scale bar, 100 μm. ( J ) Ex vivo images of excised intestines at 6 h post-administration. Rats were orally administered six Cy5-mRNA-RNACaps (L100 coated). Color scale, 0-255 gray value. Data are presented as mean ± S.D. Dots represent individual sample replicates. Statistical significance was evaluated by one-way ANOVA with Tukey’s post hoc analysis in ( E ) and ( G ). ** P < 0.01, *** P < 0.001, **** P < 0.0001. Data in (A, B, C, E, G, H, I, J) are representative of n = 3 independent experiments. All the schematic illustrations were created using Adobe Illustrator.

Article Snippet: Rat TNF-α ELISA (438204, Biolegend), Rat IL-1 beta/IL-1F2 ELISA (DY501-05, R&D Systems), Rat IL-6 ELISA (DY506-05, R&D Systems), Rat IL-17A ELISA (437904, Biolegend), Rat JE/MCP-1/CCL2 ELISA (DY3144-05, R&D Systems).

Techniques: In Vitro, Ex Vivo, Imaging, In Vivo, Enzyme-linked Immunosorbent Assay, Concentration Assay, Western Blot, Expressing, Confocal Microscopy, Immunofluorescence, Staining, Labeling, Incubation

Journal: Science translational medicine

Article Title: Oral delivery of liquid mRNA therapeutics by engineered capsule for treatment of preclinical intestinal disease

doi: 10.1126/scitranslmed.adu1493

Figure Lengend Snippet: ( A ) Experimental timeline for oral administration of IL-10 -mRNA-RNACaps to acute colitis rat models. IL-10 -mRNA: 25 μg in 3 RNACaps per rat. Acute colitis was induced in rats by providing free access to drinking water supplemented with 6.0% (w/w) dextran sulfate sodium (DSS) for 8 days. Rats were treated with RNACaps on day 2, 5 and 8. ( B and C ) Relative body weight (B) and disease activity index (DAI) (C) of healthy (plain water-treated), DSS-treated or DSS plus IL-10 -mRNA-RNACap-treated rats were monitored daily. ( D ) Quantification of colon length on day 8. ( E to J) Colon tissue protein expression of IL-10 (E), tumor necrosis factor-alpha (TNF-α) (F), interleukin-1β (IL-1β) (G), IL-6 (H), IL-17A (I) and monocyte chemoattractant protein-1 (MCP-1) (J) by ELISA. ( K to P ) Quantification of protein expression in blood of IL-10 (K), TNF-α (L), IL-1β (M), IL-6 (N), IL-17A (O) and MCP-1 (P) by ELISA. ( Q ) H&E staining images of the colon tissue sections. Dashed box indicates inset. Scale bars, 500 μm and 400 μm. Data are presented as mean ± S.D. Dots represent individual sample replicates. Statistical significance was evaluated by one-way ANOVA with Tukey’s post hoc analysis in (B to P). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. n = 5 animals per group for all panels. All the schematic illustrations were created using Adobe Illustrator.

Article Snippet: Rat TNF-α ELISA (438204, Biolegend), Rat IL-1 beta/IL-1F2 ELISA (DY501-05, R&D Systems), Rat IL-6 ELISA (DY506-05, R&D Systems), Rat IL-17A ELISA (437904, Biolegend), Rat JE/MCP-1/CCL2 ELISA (DY3144-05, R&D Systems).

Techniques: Activity Assay, Expressing, Enzyme-linked Immunosorbent Assay, Staining

Journal: Science translational medicine

Article Title: Oral delivery of liquid mRNA therapeutics by engineered capsule for treatment of preclinical intestinal disease

doi: 10.1126/scitranslmed.adu1493

Figure Lengend Snippet: ( A ) Experimental timeline for the oral administration of IL-10 -mRNA-RNACaps in acute colitis rat models. Rats were given free access to drinking water supplemented with 8.0% (w/w) DSS for 10 days to induce colitis. Afterward, plain water was provided, and rats were treated with RNACaps ( IL-10 -mRNA: 25 μg in 3 RNACaps per rat.) on days 11, 14 and 17 or sulfasalazine (SSZ, standard therapy, 100 mg/kg/day) daily. ( B and C ) Relative body weight (B) and DAI (C) of healthy (plain water-treated), DSS-treated, DSS plus IL-10 -mRNA-RNACap-treated or DSS plus SSZ-treated rats were monitored daily. ( D ) Quantification of colon length on day 17. ( E to J ) Quantification tissue protein expression of IL-10 (E), TNF-α (F), IL-1β (G), IL-6 (H), IL-17A (I) and MCP-1 (J) by ELISA. ( K to P ) Quantification of protein expression in blood of IL-10 (K), TNF-α (L), IL-1β (M), IL-6 (N), IL-17A (O) and MCP-1 (P) by ELISA. ( Q ) H&E staining images of the corresponding colon tissue sections. Dashed box indicates inset. Scale bars, 100 μm and 400 μm. Data are presented as mean ± S.D. Dots represent individual sample replicates. Statistical significance was evaluated by one-way ANOVA with Tukey’s post hoc analysis in (B to P). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. n = 5 animals per group for all panels. All the schematic illustrations were created using Adobe Illustrator.

Article Snippet: Rat TNF-α ELISA (438204, Biolegend), Rat IL-1 beta/IL-1F2 ELISA (DY501-05, R&D Systems), Rat IL-6 ELISA (DY506-05, R&D Systems), Rat IL-17A ELISA (437904, Biolegend), Rat JE/MCP-1/CCL2 ELISA (DY3144-05, R&D Systems).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Staining

Journal: Science translational medicine

Article Title: Oral delivery of liquid mRNA therapeutics by engineered capsule for treatment of preclinical intestinal disease

doi: 10.1126/scitranslmed.adu1493

Figure Lengend Snippet: ( A ) Experimental timeline for oral administration of IL-10 -mRNA-RNACaps to rats for safety assessment. IL-10 -mRNA: 25 μg in 3 RNACaps per rat. ( B and C ) Analysis of blood chemistry, including aminotransferase (ALT), aspartate aminotransferase (AST), and blood urea nitrogen (BUN) and complete blood count analysis, including white blood cell count (WBC), neutrophil (NE) (B), lymphocyte (LY), red blood cell count (RBC), hemoglobin (Hb), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and platelets (PLT) (C). ( D ) The rats were euthanized at the end of the study, and the indicated organs were sectioned and stained with H&E. Scale bars, 100 μm and 400 μm. ( E and F ) Cytokine concentration in the serum 6 h following oral administration of Fluc -mRNA-RNACap ( Fluc -mRNA: 25 μg in 3 RNACaps per rat) to healthy rats were measured using ELISA and Luminex. Cytokines measured include IL-1ra, IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, and IL-10 (E) or IL-12p70, IL-13, IL-17A, IL-18, IFN-γ, TNF-α, GM-CSF, and VEGF (F). Data are presented as mean ± S.D. Dots represent individual sample replicates. Statistical significance was evaluated by unpaired two-tailed Student’s t-test in (B, C, E, F). * P < 0.05, *** P < 0.001. n = 3 animals per group for all panels. All the schematic illustrations were created using Adobe Illustrator.

Article Snippet: Rat TNF-α ELISA (438204, Biolegend), Rat IL-1 beta/IL-1F2 ELISA (DY501-05, R&D Systems), Rat IL-6 ELISA (DY506-05, R&D Systems), Rat IL-17A ELISA (437904, Biolegend), Rat JE/MCP-1/CCL2 ELISA (DY3144-05, R&D Systems).

Techniques: In Vivo, Cell Characterization, Concentration Assay, Staining, Enzyme-linked Immunosorbent Assay, Luminex, Two Tailed Test