Journal: bioRxiv

Article Title: Integration of Patient-Derived Organoids and Organ-on-Chip Systems: Investigating Colorectal Cancer Invasion within the Mechanical and GABAergic Tumor Microenvironment

doi: 10.1101/2023.09.14.557797

Figure Lengend Snippet: (A.) Schematic representing the initial events of the metastatic cascade that can be measured using the CRC-OOC. Tumor cells in the top channel (1) can be visualized and analyzed separately from tumor cells that have invaded and adhered in the endothelial compartment (2). Additionally, tumor cells that are found in the endothelial effluent (3; circulating tumor cells (“CTC-like” cells)) can also be collected and analyzed. (B.) Circulating tumor cells were collected from the endothelial effluent of stretched and not stretched HCT116 CRC-Chips and RNAseq was performed. GO Pathway analysis was performed on a subset of genes with either a 2-fold difference between stretched and not stretched CTCs or an FDR-adjusted p-value <0.1. N=2 biological replicates with 3 pooled chips in each replicate. (C.) Gene expression of neurotransmitter-related genes were measured by a neurotransmitter-specific PCR array. HCT116 tumor cells of stretched and not stretched chips were harvested on day 6 from the top epithelial channel and isolated via FACs. Data is displayed as the gene expression fold change of stretch versus not stretch conditions Expression was normalized to the average of 5 housekeeping genes. Genes that had a 1.5-fold increase or decrease in the stretched condition are displayed. N=3 biological replicates with 3 chips pooled per biological replicate. (D.) Schematic of the production of GABA from glutamate by the enzyme GAD1. In this figure, experiments related to GABA are indicated in purple and experiments related to GAD1 are indicated in light blue. (E.) Representative confocal immunofluorescent images of the epithelial (top; 1) or endothelial (bottom; 2) channel of the CRC-Chips stained for GABA (purple) on day 6. Invaded HCT116 H2B-GFP stain positive for GABA, while HCT116 H2B-GFP tumor cells that are in the top channel stain weakly for GABA. DAPI stains the nuclei of Caco2 C2BBe1 cells in the top channel and endothelial cells in the bottom channel. Scale bars represent 200 μm in the top channel image and 100 μm in the bottom channel images. Top channel images are maximum projections that span a 35 μm Z-height with a 5 μm step size. Bottom channel images are maximum projections that span a 10 μm Z-height with a 5 μm step size. (F.) RNAseq analysis was performed on CRC organoids and normalized GAD1 expression is shown. N=5 independent donors with 2-3 replicates each. Individual data points are shown and mean ± SEM is displayed. Analysis between US and UP data was performed using an unpaired t-test; ***p<0.001 ( G.) CRC organoids were isolated from stretched chips and qPCR analysis of GAD1 gene expression was performed. N=5 independent doners with 3 replicates each. Individual data points are shown and mean ± SEM is displayed. Analysis between US and UP data was performed using an unpaired t-test p<0.05. ( H.) GAD1 mRNA expression from TCGA in KRAS, NRAS, or BRAF mutant primary colon cancer tumors. N=196 patients with KRAS, NRAS, or BRAF mutant tumors; N=201 patients with KRAS, NRAS, or BRAF wildtype tumors. Individual data points are shown and median with interquartile range is represented. Data was analyzed with an unpaired t-test; ****p<0.0001. ( I.) Kaplan-Meier curve with univariate analysis of the survival of patients with KRAS, NRAS, or BRAF mutated CRC tumors based on high versus low expression of GAD1 (defined as above or below the median GAD1 mRNA expression z-score of 0.3). Data was extracted from the TCGA. N=254 patients. Data was analyzed using a log-rank (Mantel-Cox test). ( J.) Effluent from the epithelial channel of the patient-derived organoids was collected on day 0 (D0) and day 6 (D6). GABA intensity was analyzed from extracted metabolites N=6 chips per timepoint per patient; n=4 on D0 and 2 on D6 for UK. Data was analyzed using a two-way ANOVA; ***p<0.001; ****p<0.0001. ( K .) US-H2B-GFP (top) and UP-H2B-GFP (bottom) stretched tumor-chips were stained for GABA (purple). Scale bars represent 200 μm. L. Representative 10x immunofluorescence images of the 5 tumors stained for EpCAM (green), CK20 (red), and GABA (purple). Scale bars represent 500 μm and 200 μm for UK. All schematics were made in or are from BioRender.

Article Snippet: ABAT human shRNA lentiviral particles (Santa Cruz Biotechnology, Inc., #sc-93288-V) and control shRNA lentiviral particles (Santa Cruz Biotechnology, Inc., #sc-108080) were used to produce HCT116 H2B-GFP ABAT knock down cells at a multiplicity of infection (MOI) of 5 and 2 mg/mL of puromycin (Santa Cruz Biotechnology, #sc-108071) was used to select for knocked down cells.

Techniques: Gene Expression, Isolation, Expressing, Staining, Mutagenesis, Derivative Assay, Immunofluorescence

Journal: bioRxiv

Article Title: Integration of Patient-Derived Organoids and Organ-on-Chip Systems: Investigating Colorectal Cancer Invasion within the Mechanical and GABAergic Tumor Microenvironment

doi: 10.1101/2023.09.14.557797

Figure Lengend Snippet: (A.) Invasion of HCT116 tumor-chips in the presence or absence of exogenous GABA (flowed through the epithelial channel) was measured on day 6 (D6) of the experiment and normalized to day 0 (D0) invasion. N=6 chips. Individual data are shown, with mean ± SEM represented and analyzed using a one-way ANOVA; ****p<0.0001. (B.) Intracellular [ 13 C 4 ]GABA or unlabeled GABA was measured via mass spectrometry-based metabolomics in the HCT116 tumor-chips after the addition of exogenous GABA for six days. N=3 chips. (C.) Schematic of GABA catabolism by ABAT, subsequent entry into the TCA cycle, and inhibition of ABAT activity by vigabatrin. In this figure, experiments related to GABA are indicated as purple, and experiments related to ABAT are indicated as teal. (D.) Western blot analysis of ABAT in shRNA control or ABAT shRNA HCT116 tumor cells. Cropped western blot (left) and quantification (right) confirm knockdown of ABAT. (E.) Growth rate of ABAT-knockdown or control HCT116 tumor cells when grown in traditional cell culture methods. N=3. Individual data are shown and mean ± SEM are represented. Data was analyzed using a t-test; **p<0.01. (F.) Numbers of ABAT-knockdown or control HCT116 tumor cells in the top channel on-chip as measured via fluorescence microscopy and quantified on day 0 (D0) and day 6 (D6). N=5-6 chips. Individual data are shown and mean ± SEM are represented. Data was analyzed using a two-way ANOVA; *p<0.05;***p<0.001. (G.) Invasion of ABAT knockdown (KD) or control shRNA HCT116 tumor-chips in the presence or absence of stretching was measured on day 6 (D6) of the experiment and normalized to day 0 (D0) invasion. N=5-6 chips. Individual data points are shown and mean ± SEM are represented. Data was analyzed using a one-way ANOVA; *p<0.05. (H.) Numbers of HCT116 tumor cells in the top channel on-chip in the presence or absence of stretching, with or without vigabatrin was measured via fluorescence microscopy and quantified on day 0 (D0) and day 6 (Day 6). Individual data are shown and mean ± SEM are represented. N=4 chips. Data was analyzed using a two-way ANOVA; **p<0.01. (I.) Invasion of HCT116 tumor-chips in the presence or absence of stretching, with or without vigabatrin was measured on day 6 (D6) of the experiment and normalized to day 0 (D0) invasion. N=4 chips. Individual data points are shown and mean ± SEM are represented. Data was analyzed using a one-way ANOVA; ***p<0.001. (J.) Numbers of US-H2B-GFP (red) or UP-H2B-GFP (blue) tumor cells in the top channel on-chip in the presence or absence of stretching, with or without vigabatrin was measured via fluorescence microscopy and quantified on day 0 (D0) and day 6 (Day 6). N=4-5 chips. Individual data are shown and mean ± SEM are represented. Data was analyzed using a two-way ANOVA; ns=p>0.05. (K.) Invasion of US-H2B-GFP or UP-H2B-GFP organoid-tumor chips in the presence or absence of stretching, with or without vigabatrin was measured on day 6 (D6) of the experiment and normalized to day 0 (D0) invasion. N=4-5 chips. Individual data are shown and mean ± SEM are represented. Data was analyzed using a one-way ANOVA; **p<0.01. All schematics were made in or are from BioRender.

Article Snippet: ABAT human shRNA lentiviral particles (Santa Cruz Biotechnology, Inc., #sc-93288-V) and control shRNA lentiviral particles (Santa Cruz Biotechnology, Inc., #sc-108080) were used to produce HCT116 H2B-GFP ABAT knock down cells at a multiplicity of infection (MOI) of 5 and 2 mg/mL of puromycin (Santa Cruz Biotechnology, #sc-108071) was used to select for knocked down cells.

Techniques: Mass Spectrometry, Inhibition, Activity Assay, Western Blot, shRNA, Control, Knockdown, Cell Culture, Fluorescence, Microscopy

Journal: Journal of Advanced Research

Article Title: Synergistic potentiation of the anti-metastatic effect of a Ginseng - Salvia miltiorrhiza herbal pair and its biological ingredients via the suppression of CD62E-dependent neutrophil infiltration and NETformation

doi: 10.1016/j.jare.2024.10.036

Figure Lengend Snippet: Ginseng and Salvia miltiorrhiza modulated the neutrophil-associated TIME in the lungs. (A) Experimental setup. The mice were constructed as experimental metastasis models after a period of acclimatization. The mice were prepared as experimental metastasis models following an acclimatization period. Ginseng and Salvia miltiorrhiza extracts were administered daily to the relevant groups according to the schematic diagram, and lung tissues were collected on day 28. (B) Representative bioluminescence images of metastasis in the intravenously implanted LLC mice. (C) Representative H&E images of the tumor area in the lungs of the mice (scale bar: 50 μm). (D) Quantitative analysis of the lung tumor area ( n = 3). (E) Quantitative analysis of the number of lung metastatic nodules in tumor-bearing mice( n = 15). (F) Metastasis rates of LLC tumor-bearing mice ( n = 15). (G) GOBP analysis of intersecting genes in the control, model and G + S groups. (H) GSEA of genes related to neutrophil migration. (I) Representative images of immunohistochemical staining and quantitative analysis of Ly6G in the lungs ( n = 3; scale bar: 50 μm). (J) Representative images of immunofluorescence staining and quantitative analysis of Ly6G in the lungs ( n = 3; scale bar: 100 μm). All the data are presented as mean ± SEM. #### p < 0.0001 vs. the control group. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs the model group.

Article Snippet: Human neutrophils were isolated from the peripheral blood of healthy subjects via a neutrophil isolation kit (P9040; Solarbio, Beijing, China).

Techniques: Construct, Control, Migration, Immunohistochemical staining, Staining, Immunofluorescence

Journal: Journal of Advanced Research

Article Title: Synergistic potentiation of the anti-metastatic effect of a Ginseng - Salvia miltiorrhiza herbal pair and its biological ingredients via the suppression of CD62E-dependent neutrophil infiltration and NETformation

doi: 10.1016/j.jare.2024.10.036

Figure Lengend Snippet: CPT inhibited CD62E–mediated neutrophil adhesion and recruitment to metastases. (A) Representative images and quantification of HL–60 cell adhesion to HUVECs (scale bar: 200 μm). (B) Schematic diagram of the parallel plate flow chamber combined with the microscope. (C) Number of neutrophils firmly adhered to HUVECs. (D) Representative immunofluorescence images and quantification of CD62E in the lungs ( n = 3; scale bar: 50 μm). (E) Representative western blot images of CD62E levels in the lungs and quantitative data ( n = 3). GAPDH was used as a loading control. (F) The mRNA expression levels of TNF–α, ICAM1, and VCAM1 in lung tissues were determined by qRT–PCR ( n = 3). (G) The correlation between CD62E expression and neutrophil infiltration in LUAD and LUSC was analyzed via TIMER. (H) Representative images and quantification of Ly6G + neutrophils in lungs detected by flow cytometry ( n = 4). (I) Representative immunofluorescence images and quantification of Ly6G + neutrophils in the lungs (n = 3; scale bar: 50 μm). All the data are presented as mean ± SEM. ## p < 0.01, ### p < 0.001, #### p < 0.0001 vs. the control group. * p < 0.05, ** p < 0.01, **** p < 0.0001 vs. the model group.

Article Snippet: Human neutrophils were isolated from the peripheral blood of healthy subjects via a neutrophil isolation kit (P9040; Solarbio, Beijing, China).

Techniques: Microscopy, Immunofluorescence, Western Blot, Control, Expressing, Quantitative RT-PCR, Flow Cytometry

Journal: Journal of Advanced Research

Article Title: Synergistic potentiation of the anti-metastatic effect of a Ginseng - Salvia miltiorrhiza herbal pair and its biological ingredients via the suppression of CD62E-dependent neutrophil infiltration and NETformation

doi: 10.1016/j.jare.2024.10.036

Figure Lengend Snippet: Neutrophil RNA sequencing analysis and correlation analysis of NETs and lung cancer. (A) Schematic diagram of neutrophil isolation and RNA sequencing from mouse lung tissues. (B-C) Heatmap analysis of neutrophil-related genes and KEGG analysis of altered genes. (D) Results of partial GOBP analysis of intersecting genes in the control, model and G + S groups. (E) The expression of HIST3H3 in different cancers and adjacent normal tissues was analyzed via TIMER. (F–H) The correlation between copy number alterations in HIST3H3, ELANE, and MPO and neutrophil infiltration was analyzed via TIMER. (I) The correlations between cumulative survival and HIST3H3, ELANE, MPO and neutrophil infiltration in lung cancer patients were analyzed via TIMER. A two-sided Wilcoxon rank-sum test was used. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. the control group.

Article Snippet: Human neutrophils were isolated from the peripheral blood of healthy subjects via a neutrophil isolation kit (P9040; Solarbio, Beijing, China).

Techniques: RNA Sequencing, Isolation, Control, Expressing

Journal: Journal of Advanced Research

Article Title: Synergistic potentiation of the anti-metastatic effect of a Ginseng - Salvia miltiorrhiza herbal pair and its biological ingredients via the suppression of CD62E-dependent neutrophil infiltration and NETformation

doi: 10.1016/j.jare.2024.10.036

Figure Lengend Snippet: Ginseng and Salvia miltiorrhiza reduced the generation of NETs in lung tissues, and effective substances from ginseng were screened. (A) Representative immunohistochemical images of MPO expression in the lungs (scale bar: 100 μm). (B) Representative immunofluorescence images of H3cit and NE in the lungs (scale bar: 100 μm). (C–E) Quantitative analysis of MPO, H3cit and NE expression in the lungs ( n = 3). (F) Neutrophil MPO release was detected by ELISA. The chemical structure of Rg1 is shown on the right. (G) Representative images and quantification of extracellular DNA release after Sytox™ Green staining (scale bar: 50 μm). All the data are presented as mean ± SEM. ####p < 0.0001 vs. the control group. * p < 0.05, ** p < 0.01, ***p < 0.001, **** p < 0.0001 vs. the model group.

Article Snippet: Human neutrophils were isolated from the peripheral blood of healthy subjects via a neutrophil isolation kit (P9040; Solarbio, Beijing, China).

Techniques: Immunohistochemical staining, Expressing, Immunofluorescence, Enzyme-linked Immunosorbent Assay, Staining, Control

Journal: Journal of Advanced Research

Article Title: Synergistic potentiation of the anti-metastatic effect of a Ginseng - Salvia miltiorrhiza herbal pair and its biological ingredients via the suppression of CD62E-dependent neutrophil infiltration and NETformation

doi: 10.1016/j.jare.2024.10.036

Figure Lengend Snippet: Rg1 reduced NET formation by inhibiting ROS production and ERK1/2 and MAPK phosphorylation. (A) Representative immunofluorescence images of the NET components H3cit and NE in neutrophils ( n = 3; scale bar: 50 μm). (B–C) Representative western blot images of H3cit expression in neutrophils and quantitative data. GAPDH was used as a loading control ( n = 3). (D-E) ROS production in dHL-60 cells (D) and neutrophils (E) was assessed by flow cytometry. The median fluorescence intensity of ROS was used for quantification. (F-G) Representative western blot images and quantification of p-ERK1/2, ERK1/2, p-MAPK and MAPK expression in dHL-60 cells (F) and neutrophils (G). GAPDH was used as a loading control. All the data are presented as mean ± SEM. # p < 0.05, ### p < 0.001, #### p < 0.0001 vs. the control group. * p < 0.05, ** p < 0.01, **** p < 0.0001 vs. the model group.

Article Snippet: Human neutrophils were isolated from the peripheral blood of healthy subjects via a neutrophil isolation kit (P9040; Solarbio, Beijing, China).

Techniques: Phospho-proteomics, Immunofluorescence, Western Blot, Expressing, Control, Flow Cytometry, Fluorescence

Journal: Journal of Advanced Research

Article Title: Synergistic potentiation of the anti-metastatic effect of a Ginseng - Salvia miltiorrhiza herbal pair and its biological ingredients via the suppression of CD62E-dependent neutrophil infiltration and NETformation

doi: 10.1016/j.jare.2024.10.036

Figure Lengend Snippet: Rg1 reversed NET-induced invasion, EMT, and adhesion of LLC cells and increased vascular permeability. (A) LLC cell proliferation was detected via a CCK8 assay. (B) Schematic diagram of NET isolation and coculture with LLC cells in the transwell system. (C) Images of the transwell invasion assay (scale bar, 200 μm). The number of infiltrated cells and the OD values were used for quantification. (D-E) Representative immunofluorescence images and quantification of MMP9 in the lungs (n = 3, scale bar 50 μm). (F) Representative immunofluorescence images of E-cadherin and N-cadherin in LLC cells treated with or without Rg1 or NETs. (G-I) Representative immunofluorescence images and quantification of E-cadherin and N-cadherin in the lungs ( n = 3, scale bar 50 μm). (J) Schematic diagram of the adhesion assay of LLC cells by NETs. (K) Representative fluorescence images of the adhesion assay for DiI-labeled LLC cells trapped within PMA- or Rg1-treated neutrophils. (L) RNA-seq analysis of neutrophils. GSEA of genes related to the regulation of vascular permeability. (M) Schematic diagram of the endothelial permeability assay. (N) The effects of Rg1 or NETs on vascular permeability were tested via an endothelial permeability assay. (O-P) Representative immunofluorescence images and quantification of CD31 and FITC-dextran in the lungs ( n = 3; scale bar, 50 μm). All the data are presented as mean ± SEM. # p < 0.05, #### p < 0.0001 vs. the control group. * p < 0.05, ** p < 0.01, **** p < 0.0001 vs. the model group.

Article Snippet: Human neutrophils were isolated from the peripheral blood of healthy subjects via a neutrophil isolation kit (P9040; Solarbio, Beijing, China).

Techniques: Permeability, CCK-8 Assay, Isolation, Transwell Invasion Assay, Immunofluorescence, Cell Adhesion Assay, Fluorescence, Labeling, RNA Sequencing, 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 (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: JCI insight

Article Title: Silencing SMOC2 ameliorates kidney fibrosis by inhibiting fibroblast to myofibroblast transformation.

doi: 10.1172/jci.insight.90299

Figure Lengend Snippet: Figure 1. SMOC2 is highly upregulated in mice and humans with kidney fibrosis. Quantitative immunostaining for SMOC2 (red) and αSMA (green) was performed on kidney sections obtained from mice at day 7 following (A) unilateral ureteral obstruction (UUO) or (B) folic acid injection (FA) (n = 5; 20× magnification). Scale bar: 50 μm. For the UUO model, contralateral kidney (CoK) tissue from day 14 was also included. Bottom panel in A shows magnified images taken at 40× from the selected regions of the middle row. Scale bar: 30 μm. Relative quantitation of SMOC2 and αSMA immunofluorescence, as represented in a box plot, was performed using representative images of 5 visual fields for each tissue analyzed. (C and D) Representative Western blot (n = 5/condition; Supplemental Figure 1, B and C [UUO and FA, respectively]) of SMOC2, αSMA, collagen 1α1, and fibronectin expression using kidney samples obtained from mice subjected to 7 and 14 days of UUO or FA. (E) Quantitative immunostaining for SMOC2 (red) and αSMA (green) in human kidneys with pathological fibrosis underlying chronic kidney disease (CKD) (n = 5) and nonfibrotic patients (n = 5; 20× magnification). Yellow scale bars: 50 μm. Bottom panel shows magnified images taken at 60× from the selected regions of the middle row. White scale bars: 25 μm. Relative quantitation of SMOC2 and αSMA immunofluorescence as represented in a box plot was performed using representative images of 5 visual fields for each tissue analyzed. (F) Urinary levels of SMOC2 and kidney injury molecule-1 (KIM-1) normalized to urinary creatinine were measured in patients with CKD (n = 13) compared with healthy volunteers (n = 13). Box plots describe the median (line within box), upper and lower quartiles (bounds of box), and minimum and maximum values (bars). *P < 0.05 determined by t test. Yellow arrows, tubules. White arrows, interstitium.

Article Snippet: For antibody competition studies, recombinant SMOC2 was preincubated with human SMOC2 antibody (R&D Systems, MAB5140) or human/mouse SMOC2 antibody (R&D Systems, AF5140) and placed on rotation at room temperature for 1 hour, prior to being treated on cells.

Techniques: Immunostaining, Injection, Quantitation Assay, Immunofluorescence, Western Blot, Expressing

Journal: JCI insight

Article Title: Silencing SMOC2 ameliorates kidney fibrosis by inhibiting fibroblast to myofibroblast transformation.

doi: 10.1172/jci.insight.90299

Figure Lengend Snippet: Figure 2. SMOC2-overexpressing mice are more susceptible to kidney fibrosis than WT mice. (A) Confirmation of SMOC2 overexpression in SMOC2 transgenic (SMOC2 Tg) mice by PCR (above, primers specific to recognize Tg insert) and Western blotting (below) (Supplemental Figure 3A). (B) Repre- sentative Western blot (n = 5/condition; Supplemental Figure 3B and Supplemental Figure 5B) of αSMA, collagen 1α1, fibronectin, and SMOC2 expression using kidney samples obtained from SMOC2 Tg and WT mice subjected to 7 and 14 days of unilateral ureteral obstruction (UUO). (C) Representative images of immunofluorescent staining for αSMA in CoK and fibrotic kidneys from WT and SMOC2 Tg mice at day 7 following UUO (n = 5/condition, 5 visual fields/ tissue). (D) Representative Western blot (n = 5/condition; Supplemental Figure 3C and Supplemental Figure 6B) of αSMA, collagen 1α1, fibronectin, and SMOC2 expression using kidney samples obtained from SMOC2 Tg and WT mice subjected to 7 and 14 days of folic acid (FA). (E) Representative images of immunofluorescent staining for αSMA of normal and fibrotic kidneys from WT and SMOC2 Tg mice at day 7 following FA (n = 5/condition, 10 visual fields/ tissue). (F) Representative images of picrosirius red (n = 5/condition, 10 visual fields/tissue) and Masson’s trichrome (n = 5/condition, 5 visual fields/ tissue) staining of CoK versus 7 and 14 day UUO–treated kidneys. (G) Representative images of picrosirius red and Masson’s trichrome staining of normal versus 7 and 14 day FA–treated kidneys (n = 5/condition, 5 visual fields/tissue). Confocal and light microscopy images are 20× magnification. Scale bars: 50 μM. Relative quantifications of images are represented as box plots, which describe the median (line within box), upper and lower quartiles (bounds of box), and minimum and maximum values (bars). *P < 0.05 (CoK [UUO] or Normal [FA]) and #P < 0.05 (WT at respective time point) determined by one-way ANOVA with Tukey post-hoc analysis.

Article Snippet: For antibody competition studies, recombinant SMOC2 was preincubated with human SMOC2 antibody (R&D Systems, MAB5140) or human/mouse SMOC2 antibody (R&D Systems, AF5140) and placed on rotation at room temperature for 1 hour, prior to being treated on cells.

Techniques: Over Expression, Transgenic Assay, Western Blot, Expressing, Staining, Light Microscopy

Journal: JCI insight

Article Title: Silencing SMOC2 ameliorates kidney fibrosis by inhibiting fibroblast to myofibroblast transformation.

doi: 10.1172/jci.insight.90299

Figure Lengend Snippet: Figure 3. SMOC2 induces a fibroblast-to-myofibroblast transition. (A) RNAseq was performed using kidneys from SMOC2 Tg and WT mice at day 7 following UUO treatment. REVIGO treemap visualizations are shown for enriched gene ontology (GO) categories. Highly similar GO terms for cellular components are grouped and visualized by different colors and sizes of the rectangles using semantic similarity and enrichment P values. Representative Western blots of αSMA, collagen 1α1, and fibronectin from serum-deprived primary human kidney fibroblasts (B, n = 3/condition; Supplemental Figure 8C) and NIH3T3 fibroblasts (C, n = 3/condition; Supplemental Figure 8D) treated with 10 ng/ml SMOC2 with/without TGFβ1. (D) After 1 hour of antibody pretreatment, SMOC2 or TGFβ1 was treated to serum-deprived NIH3T3 cells for 24 hours and then tested for conventional fibrotic markers, while integrin β1 antibody was pretreated with NIH3T3 cells and then treated with SMOC2 (n = 3/condition; Supplemental Figure 8E). (E) NIH3T3 fibroblasts were transfected with SMOC2-MYC, empty vector control, or negative control MGP-MYC and then immunoprecipitated with a MYC (above) or integrin antibody (below). Western blots are representative immunoprecipitation experi- ments. (F) Representative Western blot for Phospho-Focal Adhesion Kinase (P-FAK) Y925, P-Myosin Light Chain (MLC) Ser19, and P-Paxillin Tyr118 from NIH3T3 cells treated with 10 ng/ml SMOC2 or 5 ng/ml TGFβ1 for 60 minutes (n = 5/condition; Supplemental Figure 8H). (G) Phalloidin staining of F-actin after NIH3T3 cells were treated 24 hours with 10 ng/ml SMOC2 or 5 ng/ml TGFβ1 (n = 3). Top row 40×, 25 μM. Bottom row 100×, 15 μM. Box plots describe the median (line within box), upper and lower quartiles (bounds of box), and minimum and maximum values (bars). *P < 0.05 determined by t test.

Article Snippet: For antibody competition studies, recombinant SMOC2 was preincubated with human SMOC2 antibody (R&D Systems, MAB5140) or human/mouse SMOC2 antibody (R&D Systems, AF5140) and placed on rotation at room temperature for 1 hour, prior to being treated on cells.

Techniques: Western Blot, Transfection, Plasmid Preparation, Control, Negative Control, Immunoprecipitation, Staining

Journal: JCI insight

Article Title: Silencing SMOC2 ameliorates kidney fibrosis by inhibiting fibroblast to myofibroblast transformation.

doi: 10.1172/jci.insight.90299

Figure Lengend Snippet: Figure 4. SMOC2 induces the properties of myofibroblast activities. (A) REVIGO treemap visualization for highly similar GO terms describing biological processes significantly different between SMOC2 Tg and WT mice at day 7 following UUO treatment. (B) Scratch assay performed on NIH3T3 cells treated 24 hours with 10 ng/ml SMOC2. Healing percentage represented in box plot (n = 5, 3 visual fields/condition; 10× magnification, scale bar: 50 μM). (C) Boyden chamber assay performed on NIH3T3 cells treated 24 hours with 10 ng/ml SMOC2. (D) NIH3T3 cells were treated 24 hours with/without 10 ng/ml SMOC2, and then trypsinized and reseeded. After 1 hour, unattached cells were washed and cell numbers were quantified for adherence (n = 3). (E) Meta- bolic activity of control and 10 ng/ml SMOC2-treated NIH3T3 cells were measured over time by MTT assay (n = 5). (F) NIH3T3 fibroblasts were treated 24 hours with/without 10 ng/ml SMOC2, and cell proliferation was assessed by EdU labeling and FACS (n = 5). Box plots describe the median (line within box), upper and lower quartiles (bounds of box), and minimum and maximum values (bars). *P < 0.05 determined by t test.

Article Snippet: For antibody competition studies, recombinant SMOC2 was preincubated with human SMOC2 antibody (R&D Systems, MAB5140) or human/mouse SMOC2 antibody (R&D Systems, AF5140) and placed on rotation at room temperature for 1 hour, prior to being treated on cells.

Techniques: Wound Healing Assay, Boyden Chamber Assay, Activity Assay, Control, MTT Assay, Labeling

Journal: JCI insight

Article Title: Silencing SMOC2 ameliorates kidney fibrosis by inhibiting fibroblast to myofibroblast transformation.

doi: 10.1172/jci.insight.90299

Figure Lengend Snippet: Figure 5. Genetic inhibition of SMOC2 limits folic acid–induced kidney fibrosis in mice. (A) Confirmation of SMOC2 deletion in SMOC2-KO mice by PCR (above, PCR primers specific to recognize knock-in insert) and Western blotting (below). (B) Representative Western blot (n = 4/group; Supplemental Fig- ure 11) of αSMA, collagen 1α1, fibronectin, and SMOC2 expression using kidney samples obtained at day 7 from SMOC2-KO and WT mice subjected to folic acid (FA) treatment. (C) Immunofluorescent αSMA staining of KO and WT kidneys at day 7 following with/out FA treatment (n = 4/group). (D) Masson’s trichrome staining of normal and FA-treated kidneys obtained at day 7 from KO and WT mice. Confocal and light microscopy images are 20× magnification. Scale bars: 50μM. Quantification of images is represented as box plots (n = 4/condition, 10 visual fields/mice), which describe the median (line within box), upper and lower quartiles (bounds of box), and minimum and maximum values (bars). *P < 0.05 (WT normal) and #P <0.05 (WT at respective treatment) determined by one-way ANOVA with Tukey post-hoc analysis.

Article Snippet: For antibody competition studies, recombinant SMOC2 was preincubated with human SMOC2 antibody (R&D Systems, MAB5140) or human/mouse SMOC2 antibody (R&D Systems, AF5140) and placed on rotation at room temperature for 1 hour, prior to being treated on cells.

Techniques: Inhibition, Knock-In, Western Blot, Expressing, Staining, Light Microscopy

Journal: JCI insight

Article Title: Silencing SMOC2 ameliorates kidney fibrosis by inhibiting fibroblast to myofibroblast transformation.

doi: 10.1172/jci.insight.90299

Figure Lengend Snippet: Figure 6. Genetic inhibition of SMOC2 limits UUO-induced kidney fibrosis in mice. (A) Representative Western blot (n = 5/group; Supplemental Figure 12) of αSMA, collagen 1α1, fibronectin, and SMOC2 expression using kidney samples obtained at day 7 from SMOC2-KO and WT mice subjected to UUO. (B) Representative images (n = 3/group; 5 visual fields for each tissue analyzed) of immunofluorescent αSMA staining of KO and WT kidneys from normal mice and day 7 UUO mice. Relative quantitation is represented in a box plot as arbitrary units. (C) Masson’s trichrome staining of normal and 7-day UUO kidneys from WT and KO mice. Images of Masson’s trichrome staining are representative of 5–10 visual fields for each tissue analyzed. Quantification is represented in a box plot as arbitrary units (mice n = 5–6, 5–10 visual fields/mice). Confocal images are 20× magnification; scale bar: 50 μM. Light micros- copy images are 20× magnification; scale bar: 50μM. Box plots describe the median (line within box), upper and lower quartiles (bounds of box), and mini- mum and maximum values (bars). *P < 0.05 (WT CoK) and #P < 0.05 (WT at respective UUO) determined by one-way ANOVA with Tukey post-hoc analysis.

Article Snippet: For antibody competition studies, recombinant SMOC2 was preincubated with human SMOC2 antibody (R&D Systems, MAB5140) or human/mouse SMOC2 antibody (R&D Systems, AF5140) and placed on rotation at room temperature for 1 hour, prior to being treated on cells.

Techniques: Inhibition, Western Blot, Expressing, Staining, Quantitation Assay

Journal: JCI insight

Article Title: Silencing SMOC2 ameliorates kidney fibrosis by inhibiting fibroblast to myofibroblast transformation.

doi: 10.1172/jci.insight.90299

Figure Lengend Snippet: Figure 7. Silencing SMOC2 reduces TGFβ1-induced fibrotic markers in vitro and folic acid–induced kidney fibrosis in mice. (A) Scheme of the exper- imental procedure for SMOC2 siRNA–transfected NIH3T3 cells. After 24 hours of treatment with SMOC2 siRNA or scrambled siRNA (ssiRNA), NIH3T3 fibroblasts were either treated with/without TGFβ1 for 24 hours. Representative Western blot (n = 3/condition; Supplemental Figure 14) was performed for SMOC2, αSMA, collagen 1α1, and fibronectin expression. (B) Scheme of the experimental procedure for SMOC2 siRNA– or ssiRNA-injected C57BL/6 mice treated with/out folic acid (FA). Mice were injected i.v. with 30 μg/200 μl of SMOC2 siRNA or ssiRNA 4 hours before and 2, 4, and 6 days after an i.p. injection of 250 mg/kg of FA. Representative Western blot (n = 5/group; Supplemental Figure 16) was performed for SMOC2, αSMA, collagen 1α1, and fibronectin. (C) Immunofluorescent αSMA staining of kidneys obtained from mice at day 7 following with/out FA either treated with ssiRNA or SMOC2 siRNA (n = 5). (D) Masson’s trichrome staining of normal and FA-treated kidneys obtained at day 7 following ssiRNA or SMOC2 siRNA administration. Confocal and light microscopy images are 20× magnification. Scale bars: 50 μM. Quantification of images is represented as a box plot (n = 5/condition, 10 visual fields/mice), which describe the median (line within box), upper and lower quartiles (bounds of box), and minimum and maximum values (bars). *P < 0.05 (ssiRNA + vehicle) and #P < 0.05 (ssiRNA respective treatment) determined by one-way ANOVA with Tukey post-hoc analysis.

Article Snippet: For antibody competition studies, recombinant SMOC2 was preincubated with human SMOC2 antibody (R&D Systems, MAB5140) or human/mouse SMOC2 antibody (R&D Systems, AF5140) and placed on rotation at room temperature for 1 hour, prior to being treated on cells.

Techniques: In Vitro, Transfection, Western Blot, Expressing, Injection, Staining, Light Microscopy

Journal: Cells

Article Title: CCL4 Affects Eosinophil Survival via the Shedding of the MUC1 N-Terminal Domain in Airway Inflammation.

doi: 10.3390/cells14010033

Figure Lengend Snippet: Figure 1. Full-length MUC1 (MUC1-FL) expression in the epithelial cells of nasal polyps. (A) Im- munofluorescence analysis of nasal polyps obtained from patients with chronic rhinosinusitis with nasal polyps (CRSwNP) with high eosinophil count (left panels; i–iii) or those with low eosinophil count (right panels; iv–vi). MUC1-FL (green), C-terminal domain (MUC1-C, red), and the nucleus (blue) are stained. Images were captured by an FV3000 confocal microscope (400× objectives). The scale bars in the bottom-right corner indicate 10 µm. (B) Correlation of MUC1-FL expression with eosinophil count in nasal polyps. MUC1-FL intensity is indicated as a ratio to epithelial cell adhesion molecule (EpCAM) (n = 39).

Article Snippet: Human eosinophils were separated with high purity (>98%) from peripheral blood obtained from healthy individuals with slightly increased eosinophils (approximately 300–500/μL) using an eosinophil isolation kit with a MACS system (Miltenyi Biotec, Bergish Gladbach, Germany).

Techniques: Expressing, Staining, Microscopy

Journal: Cells

Article Title: CCL4 Affects Eosinophil Survival via the Shedding of the MUC1 N-Terminal Domain in Airway Inflammation.

doi: 10.3390/cells14010033

Figure Lengend Snippet: Figure 2. Effect of eosinophils on full-length MUC1 (MUC1-FL) expression in airway epithelial cells. (A–D) BEAS-2B cells were coincubated overnight with purified peripheral blood eosinophils. MUC1- FL mRNA levels (A), MUC1-FL protein levels (B), and MUC1 C-terminal domain (MUC1-C) protein levels (C) were evaluated. (D) Immunofluorescence analysis of MUC1-FL (green), MUC1-C (red), and the nucleus (blue) are shown in the upper (without eosinophils) and lower (with eosinophils) panels. Images were captured by an FV3000 confocal microscope (400× objectives). Scale bars in the bottom-right corner indicate 10 µm. Results were representative of at least three experiments. (E) MUC1-FL protein expression in BEAS-2B coincubated with the supernatants of eosinophilic mucin overnight. Patients underwent endoscopic sinus surgery under general anesthesia. Mucin samples were collected from the sinuses of refractory ECRS subjects. Values in (A–C,E) represent the mean ± SEM of four experiments; # p < 0.05, ## p < 0.01 (vs. vehicle).

Article Snippet: Human eosinophils were separated with high purity (>98%) from peripheral blood obtained from healthy individuals with slightly increased eosinophils (approximately 300–500/μL) using an eosinophil isolation kit with a MACS system (Miltenyi Biotec, Bergish Gladbach, Germany).

Techniques: Expressing, Purification, Immunofluorescence, Microscopy

Journal: Cells

Article Title: CCL4 Affects Eosinophil Survival via the Shedding of the MUC1 N-Terminal Domain in Airway Inflammation.

doi: 10.3390/cells14010033

Figure Lengend Snippet: Figure 3. Relation between full-length MUC1 (MUC1-FL) and CCL4 expression in the epithelial cells of nasal polyps. (A) Immunofluorescence staining of nasal polyps obtained from patients with CRSwNP with high or low eosinophil count. MUC1-FL, CCL4, and epithelial cell adhesion molecule (EpCAM) expression levels were evaluated. MUC1-FL (pink), CCL4 (green), EpCAM (orange), MBP (red), and the nucleus (blue) are stained with hematoxylin and eosin (H&E). Images were captured by an FV3000 confocal microscope (100× objectives). The scale bars in the bottom-right corner indicate 100 µm. (B,C) Correlation of CCL4 expression with the eosinophil count in nasal polyps (B) and MUC1-FL expression (C). MUC1-FL and CCL4 intensities are indicated as a ratio to EpCAM (n = 39).

Article Snippet: Human eosinophils were separated with high purity (>98%) from peripheral blood obtained from healthy individuals with slightly increased eosinophils (approximately 300–500/μL) using an eosinophil isolation kit with a MACS system (Miltenyi Biotec, Bergish Gladbach, Germany).

Techniques: Expressing, Immunofluorescence, Staining, Microscopy

Journal: Cells

Article Title: CCL4 Affects Eosinophil Survival via the Shedding of the MUC1 N-Terminal Domain in Airway Inflammation.

doi: 10.3390/cells14010033

Figure Lengend Snippet: Figure 4. Effect of CCL4-mediated reduction of full-length MUC1 (MUC1-FL) on eosinophil survival. (A,B) BEAS-2B was stimulated overnight with recombinant human CCL4 (10 µg/mL). MUC1-FL expression (A) and matrix metalloproteases (ADAM17 and MMP14) mRNA levels (B) in BEAS-2B. (C–E) BEAS-2B and purified eosinophils were coincubated with or without anti-CCL4 neutralizing antibody (10 µg/mL). MUC1-FL protein levels in BEAS-2B (C), CCL4 concentration in supernatants of cell culture (D), and eosinophil survival (E) were evaluated. (F) Purified eosinophils were incubated overnight on a recombinant human MUC1-coated plate, followed by the evaluation of their survival. Images (MUC1-FL, green; nucleus, blue) in A were captured by an FV3000 confocal microscope (400× objectives) with scale bars (20 µm) in the bottom-right corner, which were representative of at least three experiments. The values in (B–F) represent the mean ± SEM of four experiments. # p < 0.05, ## p < 0.01 (vs. without rhCCL4 in (B), without eosinophils in (C,D), without BEAS-2B in (E), and without rhMUC1 in (F)). ** p < 0.01 (between the two groups).

Article Snippet: Human eosinophils were separated with high purity (>98%) from peripheral blood obtained from healthy individuals with slightly increased eosinophils (approximately 300–500/μL) using an eosinophil isolation kit with a MACS system (Miltenyi Biotec, Bergish Gladbach, Germany).

Techniques: Recombinant, Expressing, Purification, Concentration Assay, Cell Culture, Incubation, 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 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