Journal: Scientific reports

Article Title: ETV1 activates a rapid conduction transcriptional program in rodent and human cardiomyocytes.

doi: 10.1038/s41598-018-28239-7

Figure Lengend Snippet: Figure 2. Cardiomyocyte-specific knockout of ETV1 slows atrial and His-Purkinje system conduction. Etv1flox/

Article Snippet: P1 NRVM heart lysates were purified using Miltenyi Biotec rat neonatal cardiomyocyte isolation kit (Miltenyi Biotec, 130–105–420) according to the manufacture’s protocol.

Techniques: Knock-Out

Journal: Scientific reports

Article Title: ETV1 activates a rapid conduction transcriptional program in rodent and human cardiomyocytes.

doi: 10.1038/s41598-018-28239-7

Figure Lengend Snippet: Figure 3. Cardiomyocyte deletion of ETV1 resulted in decreased expression of fast conduction genes in atrial and His-Purkinje system (HPS) myocytes. (A) Quantitative RT-PCR of fast conduction gene RNA levels (normalized to Gapdh) comparing 10–12-week-old Etv1 WT (Etv1flox/flox) and Etv1 cKO (Etv1flox/flox, Myh6- Cre) FACS-purified ventricular, atrial, and Purkinje myocytes. Relative Nkx2–5, Gja5, and Scn5a expression displayed versus control, Etv1 WT (n = 4). (B) Immunoblot assessment of Etv1 WT and Etv1 cKO atrial tissue lysates detecting NKX2–5, Cx40, NaV1.5, and Vinculin (loading control). (C) Protein level densitometric quantification (normalized to vinculin), displayed relative to Etv1 WT (n = 5). (D) Immunofluorescence evaluation of NKX2–5, Cx40, and NaV1.5 expression in 10-week-old Etv1 WT and Etv1 cKO atria/ventricular sections. (E) Immunofluorescence evaluation of NKX2–5, Cx40, and NaV1.5 expression in 10-week-old Etv1 WT and Etv1 cKO HPS sections. Positive CNTN2 expression identified HPS cells. Nuclei were identified by DAPI (blue). LA, left atria; LV, left ventricle. Data represent mean ± SEM. *P < 0.05, 2-tailed Student’s t test. Scale bars: 50 um.

Article Snippet: P1 NRVM heart lysates were purified using Miltenyi Biotec rat neonatal cardiomyocyte isolation kit (Miltenyi Biotec, 130–105–420) according to the manufacture’s protocol.

Techniques: Expressing, Quantitative RT-PCR, Purification, Control, Western Blot, Immunofluorescence

Journal: Scientific reports

Article Title: ETV1 activates a rapid conduction transcriptional program in rodent and human cardiomyocytes.

doi: 10.1038/s41598-018-28239-7

Figure Lengend Snippet: Figure 5. ETV1 regulates the diversity of sodium channel biophysical properties between ventricular, atrial, and Purkinje myocytes. Whole-cell patch clamp data from dissociated cardiomyocytes (ventricular, right atrial, Purkinje myocytes) using 10–12 week-old Etv1 WT (Etv1flox/flox) and Etv1 cKO (Etv1flox/flox, Myh6-Cre) mice in a Cntn2-EGFP background (n = 4). (A) Comparison of sodium current–voltage (I–V) relationship. Maximum conductance was calculated to assess significant differences among experimental groups. (B) Voltage dependence of steady-state activation. Voltage at half activation (V0.5, activation) was calculated to assess significant differences among experimental groups. (C) Voltage dependence of steady-state inactivation. Voltage at half inactivation (V0.5, inactivation) was calculated to assess significant differences among experimental groups. (D) Time course of recovery from inactivation. Tau of recovery (τrecovery) was calculated to assess significant differences among experimental groups. Number of cells analyzed per cell type (ventricle, right atria, Purkinje) included in each graph legend. Patch clamp protocol diagrams are included for each endpoint. Data represent mean ± SEM. *P < 0.05, 1-way ANOVA.

Article Snippet: P1 NRVM heart lysates were purified using Miltenyi Biotec rat neonatal cardiomyocyte isolation kit (Miltenyi Biotec, 130–105–420) according to the manufacture’s protocol.

Techniques: Patch Clamp, Comparison, Activation Assay

Journal: Scientific reports

Article Title: ETV1 activates a rapid conduction transcriptional program in rodent and human cardiomyocytes.

doi: 10.1038/s41598-018-28239-7

Figure Lengend Snippet: Figure 6. ETV1-transduced neonatal rat ventricular myocytes (NRVMs) upregulates a His-Purkinje system gene signature. (A) Volcano plot of relative transcript expression from NRVMs transduced with either Ad-Etv1- EGFP or Ad-EGFP. RNA-sequencing (RNA-seq) comparison revealed a total of 9,236 differentially expressed genes (normalized counts ≥ 5, padj < 0.05). All significantly different genes (padj < 0.05) are labeled blue (downregulated) or red (enriched) and all nonsignificantly different transcripts labeled in gray. Of these there were 4,696 upregulated and 4,540 downregulated genes in Ad-Etv1-EGFP versus Ad-EGFP transduced NRVMs. (B) Functional clustering of upregulated genes in Ad-Etv1 transduced NRVMs highlighted significantly enriched ETV1-dependent cellular processes (top 20 non-redundant categories are shown). Pathways are color coded to represent genes clustered into functional classes for heat maps in C. (C) Comparative RNA-seq between 21-day-old (P21) wild-type mouse FACS-purified Purkinje cell (PC)/ventricular myocytes (VM) and Ad-Etv1-EGFP/Ad-EGFP transduced NRVMs. Heat map representation of 88 genes differentially expressed in Ad-Etv1-EGFP versus Ad-EGFP transduced NRVMs (n = 3) plotted adjacent to average fold change expression in PCs and VMs. Genes clustered into functional groups demonstrate that ETV1 regulates a PC transcriptome in neonatal cardiomyocytes.

Article Snippet: P1 NRVM heart lysates were purified using Miltenyi Biotec rat neonatal cardiomyocyte isolation kit (Miltenyi Biotec, 130–105–420) according to the manufacture’s protocol.

Techniques: Expressing, Transduction, RNA Sequencing, Comparison, Labeling, Functional Assay, Purification

Journal: Scientific reports

Article Title: ETV1 activates a rapid conduction transcriptional program in rodent and human cardiomyocytes.

doi: 10.1038/s41598-018-28239-7

Figure Lengend Snippet: Figure 8. Activation of ETV1 in human induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs) leads to increased expression of rapid conduction genes and sodium current. (A) Schematic representation of hiPSC-CM generation and maturation (day 0–21), transduction of Ad-Etv1-EGFP or Ad-EGFP (day 24), and timepoint for experimentation (day 38–40). (B) Quantitative RT-PCR analysis of Etv1, NKX2–5, GJA5, SCN5A, and MYL2 in hiPSC-CM transduced with either Ad-Etv1-EGFP or Ad-EGFP (n = 4). (C) Whole-cell patch clamp was performed on Ad-Etv1-EGFP (n = 12) or Ad-EGFP (n = 9) transduced hiPSC-CMs. Sodium current–voltage (I–V) relationship comparison. (D) hiPSC-CM NaV peak conductance (gNaV-peak). gNaV-peak following −120 mV to −35 mV depolarization step was measured for Ad-Etv1-EGFP (n = 12) or Ad-EGFP (n = 9) transduced hiPSC-CMs. Data represent mean ± SEM. *P < 0.05, 2-tailed Student’s t test.

Article Snippet: P1 NRVM heart lysates were purified using Miltenyi Biotec rat neonatal cardiomyocyte isolation kit (Miltenyi Biotec, 130–105–420) according to the manufacture’s protocol.

Techniques: Activation Assay, Derivative Assay, Expressing, Transduction, Quantitative RT-PCR, Patch Clamp, Comparison

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: eLife

Article Title: UBTD1 regulates ceramide balance and endolysosomal positioning to coordinate EGFR signaling

doi: 10.7554/eLife.68348

Figure Lengend Snippet: ( A–L ) DU145 cells were transfected for 48 hr with the indicated siRNA (control, siCTRLpool or siCTRLsingle1; control, siCTRL; UBTD1: siUBTD1pool or siUBTD1single1 or single2). ( A,C,H ) Western blotting images of phospho-kinases spotted on the Proteome Profiler Human Phospho-Kinase Array in complete media (A) or under EGF stimulation (C, 50 ng/ml, 10 min) or serum-starved ( H ). Phospho-EGFR double spots are marked in red rectangles. ( B ) Immunoblot and quantification (n = 3 independent experiments) of p-EGFR (Y1068 or Y1086). p-EGFR levels were quantified by calculating the ratio between p-EGFR and EGFR, both normalized to loading control signal. Immunoblot of UBTD1 shows the level of siRNA depletion. ( D, E ) Immunoblot and quantification (n = 3 independent experiments) of pSTAT3, p-ERK, and p-AKT. p-STAT3, p-ERK, and p-AKT levels were quantified by calculating the ratio between phospho-protein and total-protein, both normalized to loading control signal. Immunoblot of UBTD1 shows the level of siRNA depletion. ( F ) Representative wide-field immunofluorescence images (left) and quantification (right) of pSTAT3 nuclear translocation corresponding to nuclei/cytoplasm mean intensity ratio of pSTAT3. ( G ) mRNA quantification of STAT3 target genes (bcl2, mmp2, mmp9 and hif2). ( I ) Quantification of EGFR mRNA level. ( J ) EGF secretion measured by ELISA. ( K ) mRNA quantification of EGFR ligands. ( L ) EGFR ligands secretion measured by ELISA. Scale bar = 10 µm. n ≥ 3 independent experiments; ns = non-significant; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; ( F, I, J ) two-tailed t-test; ( B,C,E,G, K,L ) two-way ANOVA and Bonferroni’s multiple comparisons test; data are mean ± s.e.m. Figure 1—figure supplement 1—source data 1. Uncropped western blot for .

Article Snippet: Relative phosphorylation levels of 43 kinases and two related proteins were assessed using the Proteome Profiler Human Phospho-Kinase Array Kit (R and D Systems), according to the manufacturer’s instructions.

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

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: Nano Today

Article Title: SNA·SMNP·CBE system: A novel integrative strategy for β-hemoglobinopathies gene therapy

doi: 10.1016/j.nantod.2024.102558

Figure Lengend Snippet: Fig. 1. Schematic representation of experimental protocol and workflow of this present work. The top panel is the schematic illustration of the SNA substrates inserts into commercial dishes for CD34+HSPCs enrichment. Below which is the schematic representation of SMNP delivery CBE- and sgRNA-plasmids into commercial dishes with SNA substrates inserts. Then, after obtaining umbilical cord blood cells, SNA substrate and SMNP delivery mediated CBE base editing (SNA⋅SMNP⋅CBE)— enable efficiently and precisely modify BCL11A promoter to achieve C-T conversion and HBG elevation in CD34+HSPCs. The edited human CD34+HSPC was successively transplanted into SCID mouse by intraosseous injection to detect the CD34 engraftment.

Article Snippet: Method 2: Immno-magnetic isolation (IMS) For CD34 microbead magnetic-activated cell sorting isolation, a cell separation kit (CD34 MicroBead Kit, Miltenyi Biotec, 130–046–702) was used, as per the guided instruction.

Techniques: Injection

Journal: Nano Today

Article Title: SNA·SMNP·CBE system: A novel integrative strategy for β-hemoglobinopathies gene therapy

doi: 10.1016/j.nantod.2024.102558

Figure Lengend Snippet: Fig. 3. supramolecular nanoparticle (SMNP) enables delivery of CBE and sgRNA plasmids to modify BCL11A to achieve HBG over expression in vitro. (A), Schematic representation of base-editing experiments in human umbilical cord blood derived CD34+HSPCs enriched by SNA substrate CBE-, sgRNA-(with a red fluorescent label (mCherry) expressing plasmid were co-delivered by SMNP in human CD34+HSPCs enriched by SNA substrate. (B), Representative images of mCherry + cells at 2 h post-transfection following SMNP delivery CBE-, sgRNA-plasmids. (C), Representative flow cytometry results of edited CD34 positive cells ratio after SMNP delivery CBE- and sgRNA- plasmids. (D), Editing efficiency of targeted BCL11A PCR products was detected by Sanger sequencing, with C-T conversion value of 32.7 % base edits at position C1, and 9.5 % at position C2.(E), Real-time PCR analysis of mCherry (left panel), BCL11A (middle panel) and HBG (right panel) expression in CD34+HSPCs subjected with/without SMNP delivering co-encapsulated CBE- and sgRNA targeting BCL11A- plasmids (edited CD34+HSPCs/ naïve CD34+HSPCs). CD34+HSPCs delivered by SMNP co-encapsulated CBE- and sgRNA targeting BCL11A- plasmids was designated as edited group, and unedited group. Results are normalized to GAPDH and shown as mean±SEM. (n=3 biological replicates). Statistical tests compare edited samples and unedited control group (**P＜ 0.01). (F), Western blots were used to detect HBG expression at protein level in edited CD34+HSPCs and naïve CD34+HSPCs. Results are representative of three biological replicates.

Article Snippet: Method 2: Immno-magnetic isolation (IMS) For CD34 microbead magnetic-activated cell sorting isolation, a cell separation kit (CD34 MicroBead Kit, Miltenyi Biotec, 130–046–702) was used, as per the guided instruction.

Techniques: Over Expression, In Vitro, Derivative Assay, Expressing, Plasmid Preparation, Transfection, Flow Cytometry, Sequencing, Real-time Polymerase Chain Reaction, Control, Western Blot

Journal: Nano Today

Article Title: SNA·SMNP·CBE system: A novel integrative strategy for β-hemoglobinopathies gene therapy

doi: 10.1016/j.nantod.2024.102558

Figure Lengend Snippet: Fig. 4. Comparison of our novel system and conventional strategy in the aspect of editing efficiency and CD34+HSPCs property. (A), Editing efficiency of targeted BCL11A PCR products from CD34+HSPCs subjected to different strategies. The top panel (SNA⋅SMNP group) is CD34+HSPCs was enriched by our SNA substrate and transfected with CBE- and sgRNA targeting BCL11A- plasmids by SMNP delivery (with C-T conversion value of 32.7 % base edits at position C1, and 9.5 % at position C2). The middle panel (IMS⋅EP group) is CD34+HSPCs was isolated by conventional immunomagnetic bead method (IMS) and transfected CBE- and sgRNA targeting BCL11A- plasmids with electroporation (EP) (with C-T conversion value of 33.7 % base edits at position C1, and 10.2 % at position C2). The bottom panel (control group) was CD34+HSPCs without edited treatment. (B), Transcriptome analysis was performed in edited CD34+HSPCs with different strategies, and the control naïve CD34+HSPCs. Venn diagram of differential expression genes among the three groups: SNA⋅SMNP CD34+HSPCs, IMS⋅EP CD34+HSPCs and naïve CD34+HSPCs (left panel). Gene cluster analysis of transcriptome results among the three groups: SNA⋅SMNP treated CD34+HSPCs, IMS⋅EP treated CD34+HSPCs and naïve CD34+ (right panel). (C), CD34 mRNA expression in three groups SNA⋅SMNP CD34+HSPCs, IMS⋅EP CD34+HSPCs and naïve CD34+HSPCs. Results shown as mean±SEM (n=3 biological replicates). (D) mRNA expression of hematopoietic stem cell marker (CD133, CD90) and cell differentiation markers (GATA-1, SPI1, CD235a and CD33) in four groups SNA⋅SMNP CD34+HSPCs, IMS⋅EP CD34+HSPCs, naïve CD34+HSPCs, and PMBC. CD133 and CD90 were shown in left panel, while differ entiation markers including GATA-1, SPI1, CD235a and CD33 were presented in right panel. Results shown as mean±SEM (n=3 biological replicates).

Article Snippet: Method 2: Immno-magnetic isolation (IMS) For CD34 microbead magnetic-activated cell sorting isolation, a cell separation kit (CD34 MicroBead Kit, Miltenyi Biotec, 130–046–702) was used, as per the guided instruction.

Techniques: Comparison, Transfection, Isolation, Electroporation, Control, Quantitative Proteomics, Expressing, Marker, Cell Differentiation

Journal: Nano Today

Article Title: SNA·SMNP·CBE system: A novel integrative strategy for β-hemoglobinopathies gene therapy

doi: 10.1016/j.nantod.2024.102558

Figure Lengend Snippet: Fig. 5. Tracks of editing feasibility of SNA to maintain CD34 property by Intraosseous injection of transplanted edited CD34+HSPCs into SCID mouse. (A), Gene edited human CD34+HSPCs were transplanted into SCD mouse with two different injection strategies: intraosseous injection into the bone marrow of the mouse tibia (left panel) and tail vein injection (right panel). (B), Representative immunofluorescence images (scale bar=8μm) of CD34 in the bone marrow (left panel) and peripheral blood of mice at 14 weeks of intraosseous injection (top) and tail vein injection (below) strategy, respectively. (C), Representative flow cytometry results of CD45+ proportion in the bone marrow (left panel) and peripheral blood of transplanted mice with intraosseous injection (labeled with Red) and tail vein injection (labeled with green) strategy, respectively. Detection time was set at 10-, 12-, 14-week after transplantation. (D), Real-time PCR analysis of CD34 mRNA expression in the bone marrow (left panel) and peripheral blood of mice with intraosseous injection (labeled with Red) and tail vein injection (labeled with green) strategy, respectively. Detection time was set at 10-, 12-, 14-week after transplantation. Results shown as mean±SEM (n=3 biological replicates, *P<0.05, **P<0.01).

Article Snippet: Method 2: Immno-magnetic isolation (IMS) For CD34 microbead magnetic-activated cell sorting isolation, a cell separation kit (CD34 MicroBead Kit, Miltenyi Biotec, 130–046–702) was used, as per the guided instruction.

Techniques: Injection, Immunofluorescence, Flow Cytometry, Labeling, Transplantation Assay, Real-time Polymerase Chain Reaction, Expressing

Journal: Nano Today

Article Title: SNA·SMNP·CBE system: A novel integrative strategy for β-hemoglobinopathies gene therapy

doi: 10.1016/j.nantod.2024.102558

Figure Lengend Snippet: Fig. 6. Detection of BCL11A gene modification and HBG/HBB expression after transplantation of edited human CD34+HSPCs. (A), Representative Sanger sequencing of BCL11A from bone marrow (left panel) and peripheral blood (right panel) of transplanted mice, evidenced from C-T conversions. (B), (C), Real-time PCR was used to detect human HBG and HBB expression in the bone marrow (B) and peripheral blood (C) of SCID mice at 10, 12, 14weeks after transplantation of edited human CD34+HSPCs.

Article Snippet: Method 2: Immno-magnetic isolation (IMS) For CD34 microbead magnetic-activated cell sorting isolation, a cell separation kit (CD34 MicroBead Kit, Miltenyi Biotec, 130–046–702) was used, as per the guided instruction.

Techniques: Modification, Expressing, Transplantation Assay, Sequencing, Real-time Polymerase Chain Reaction

Journal: Nano Today

Article Title: SNA·SMNP·CBE system: A novel integrative strategy for β-hemoglobinopathies gene therapy

doi: 10.1016/j.nantod.2024.102558

Figure Lengend Snippet: Fig. 7. Comparison of our novel strategy and conventional strategy in maintain CD34 property in vivo. (A), Schematic illustration of protocol. The top panel is the process of conventional strategy IMS⋅EP⋅CBE3⋅intravenous (tail vein) injection, while the below panel is the process of our novel strategy SNA⋅SMNP⋅CBE3 ⋅intraosseous injection. After transplantation edited human CD34+HSPCs with different treatment at 14 weeks, SCID mice from the two different groups were euthanatized, mononuclear cells were resorted and enriched from the bone marrow of mice in both groups. Then, transcriptome analysis was performed to detect and compare the gene expression profiles in each group with naïve CD34 controls. (B), Representative flow cytometry results of CD45+ proportion in the bone marrow of SNA⋅SMNP⋅CBE3⋅intraosseous injection mice and IMS⋅EP⋅CBE3⋅intravenous (tail vein) injection counterparts. (C), Real-time PCR analysis of human HBG expression in the bone marrow (left panel) and peripheral blood (right panel) of SNA⋅SMNP⋅CBE3⋅intraosseous injection mice and IMS⋅EP⋅CBE3⋅ intravenous (tail vein) injection counterparts. (D), Transcriptome analysis was performed in edited CD34+HSPCs with different strategies, and the control naïve CD34+HSPCs. Venn diagram of differential expression genes among the three groups: IMS⋅EP⋅CBE3⋅intravenous, SNA⋅SMNP⋅CBE3⋅intraosseous injection and naïve CD34+HSPCs (left panel). Gene cluster analysis of transcriptome results among the three groups (right panel). (E), Representative CD34 expression by Real-time PCR in the three groups: SNA⋅SMNP⋅CBE3⋅intraosseous group, IMS⋅EP⋅CBE3⋅tail vein injection counterparts, as well as naïve CD34+HSPCs. Results shown as mean±SEM (n=3 biological replicates, **P<0.01).

Article Snippet: Method 2: Immno-magnetic isolation (IMS) For CD34 microbead magnetic-activated cell sorting isolation, a cell separation kit (CD34 MicroBead Kit, Miltenyi Biotec, 130–046–702) was used, as per the guided instruction.

Techniques: Comparison, In Vivo, Injection, Transplantation Assay, Gene Expression, Flow Cytometry, Real-time Polymerase Chain Reaction, Expressing, Control, Quantitative Proteomics

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