Journal: Nucleic Acids Research

Article Title: A regulatory circuit comprising GATA1/2 switch and microRNA-27a/24 promotes erythropoiesis

doi: 10.1093/nar/gkt848

Figure Lengend Snippet: GATA-1 was located on the upstream of miR-23a∼27a∼24-2 cluster and activated its expression during erythropoiesis. ( A ) Q-PCR analysis of the primary transcript of the miR-23a∼27a∼24-2 cluster in K562s undergoing erythroid differentiation caused by hemin treatment for 0, 24, 48 and 72 h. ( B ) Q-PCR analysis of the mature transcripts of miR-27a, miR-24 and miR-23a in K562s undergoing erythroid differentiation caused by hemin treatment for 0, 24, 48 and 72 h. ( C ) Northern blot analysis of miR-27a, miR-24 and miR-23a in K562s undergoing erythroid differentiation. U6 snRNA was used as a loading control. ( D ) Q-PCR analysis of mature miR-27a and miR-24 expression in CD34+ HPCs undergoing day 4, 7, 11, 15 and 18 of E culture. ( E ) Representation of the human −603 bp miR-23a∼27a∼24 cluster promoter fragments. ( F ) ChIP-PCR and ChIP-qPCR analysis of the GATA-1 hit on the −557 site at the miR-23a∼27a∼24 cluster promoter in K562s. ChIP-q-PCR results are shown as fold enrichment compared with input. Error bars represent the standard deviation obtained from three independent experiments. ** P < 0.01. ( G ) Immunoblot analysis of GATA-1 expression in K562s treated with siRNAs specific to GATA-1 (si_GATA-1) for 48 h or K562s transfected with a construct overexpressing GATA-1 (over_GATA-1) for 48 h, respectively. ( H ) Q-PCR analysis of Pri-27a∼24, mature miR-27a and miR-24 expression in K562s treated as described in (G). ( I ) Immunoblot analysis of GATA-1 expression in K562s undergoing erythroid differentiation. ( J ) ChIP-PCR and ChIP-q-PCR analysis of GATA-1 and pol II occupancy at the −557 site in hemin-treated K562s at 0 and 48 h. ChIP-q-PCR results are shown as fold enrichment compared with input. Error bars represent the standard deviation obtained from three independent experiments. * P < 0.05. ( K ) Functional activity of GATA-1 on the wild-type GATA site (WT) or mutant site (MUT) of the miR-23a∼27a∼24 promoter in a luciferase reporter analysis. Error bars represent the standard deviation obtained from three independent experiments. * P < 0.05.

Article Snippet: CD34+ cells were enriched from mononuclear cells through positive immunomagnetic selection (CD34 MultiSort kit, Miltenyi Biotec, Bergisch-Glad-bach, Germany).

Techniques: Expressing, Northern Blot, Control, ChIP-qPCR, Standard Deviation, Western Blot, Transfection, Construct, Functional Assay, Activity Assay, Mutagenesis, Luciferase

Journal: Nucleic Acids Research

Article Title: A regulatory circuit comprising GATA1/2 switch and microRNA-27a/24 promotes erythropoiesis

doi: 10.1093/nar/gkt848

Figure Lengend Snippet: MiR-27a and miR-24 promoted erythroid differentiation in CD34+ HPCs. ( A ) Monitoring of the GFP + population (left panel) and the CD235a stained GFP + fraction (medium panel) of Lenti-miRNA-transduced CD34+ HPCs on day 15 of E culture. The morphology (May-Grunwald Giemsa staining) of CD34+ HPCs derivatives on day 15 is shown in the right panel. A 400X magnification of a representative field is shown. ( B ) Detection of the erythroid differentiation degree of CD34+ HPCs transduced with Lenti-27a, Lenti-24 or Lenti-GFP in E culture at the indicated time. Percentages of basophilic (Bas), polychromatophilic (Pol), orthochromatic (Ort) erythroblasts and erythrocytes (Ery) were determined by May-Grunwald/Giemsa staining of cytospin preparations. ( C ) Q-PCR analysis of gamma-globin mRNA expression in CD34+ HPCs transduced with Lenti-27a, Lenti-24 or Lenti-GFP in E culture at the indicated time. ( D ) A comparison of the erythroid colony-forming capacity (CFU-E, BFU-E) of CD34+ HPCs transduced with Lenti-miRNAs. Error bars represent the standard deviation obtained from three independent experiments. * P < 0.05; ** P < 0.01. ( E ) FACS monitoring of CD34+ HPCs transduced with Zip-miRNA or Zip-GFP as described in (A). ( F ) Detection of the erythroid differentiation degree of CD34+ HPCs transduced with Zip-27a, Zip-24 or Zip-GFP as described in (B). ( G ) Detection of gamma-globin mRNA level in CD34+ HPCs transduced with Zip-miRNA. ( H ) Colony-forming assay of CD34+ HPCs transduced with Zip-miRNA as described in (D).

Article Snippet: CD34+ cells were enriched from mononuclear cells through positive immunomagnetic selection (CD34 MultiSort kit, Miltenyi Biotec, Bergisch-Glad-bach, Germany).

Techniques: Staining, Transduction, Expressing, Comparison, Standard Deviation

Journal: Nucleic Acids Research

Article Title: A regulatory circuit comprising GATA1/2 switch and microRNA-27a/24 promotes erythropoiesis

doi: 10.1093/nar/gkt848

Figure Lengend Snippet: GATA-2 was post-transcriptionally regulated by miR-27a and miR-24 during erythropoiesis. ( A ) A computer prediction of conserved and mutated binding sites within the 3′ UTR of GATA-2 mRNA for miR-27a and miR-24. ( B ) Relative luciferase activity of the indicated GATA-2 reporter constructs. Error bars represent the standard deviation obtained from three independent experiments. * P < 0.05. ( C ) Immunoblot analysis of GATA-2 in K562s transfected with scramble or miRNA mimics (miR-27a, miR-24) or inhibitors (Anti-27a, Anti-24). ( D ) Immunoblot analysis of GATA-2 in CD34+ HPCs transduced with Lenti-GFP control and lentivirus expressing miR-27a or miR-24 (Lenti-27a or Lenti-24). ( E, F ) ‘Rescue’ assays for miRNAs and GATA-2 during erythroid differentiation. Immunoblot analysis of GATA-2 in K562s treated with scramble or Anti-27a/24 (E) for 24 h. These cells were subsequently treated for another 24 h with control siRNAs or siRNAs specific to GATA-2 and were then treated with hemin treatment for 0, 48 and 72 h. (F) FACS analysis of K562s stained for CD71 and CD235a expression after 48 h of hemin induction as described earlier in the text.

Article Snippet: CD34+ cells were enriched from mononuclear cells through positive immunomagnetic selection (CD34 MultiSort kit, Miltenyi Biotec, Bergisch-Glad-bach, Germany).

Techniques: Binding Assay, Luciferase, Activity Assay, Construct, Standard Deviation, Western Blot, Transfection, Transduction, Control, Expressing, Staining

Journal: Nucleic Acids Research

Article Title: A regulatory circuit comprising GATA1/2 switch and microRNA-27a/24 promotes erythropoiesis

doi: 10.1093/nar/gkt848

Figure Lengend Snippet: The GATA switch regulated miR-27a and miR-24 expression. ( A ) ChIP-PCR and ChIP-q-PCR analysis of GATA-1, GATA-2 and Pol II occupancy at the −557 site in hemin-treated K562s at 0 and 48 h. ChIP-q-PCR results are shown as fold enrichment compared with input. Error bars represent the standard deviation obtained from three independent experiments. * P < 0.05; ** P < 0.01. ( B ) Immunoblot analysis of GATA-1 and GATA-2 expression in K562s undergoing erythroid differentiation for 0, 24, 48 and 72 h. ( C ) Functional activity of GATA-1 and GATA-2 on the wild-type GATA site (WT) or mutant site (MUT) of the miR-23a∼27a∼24 promoter in a luciferase reporter analysis. Error bars represent the standard deviation obtained from three independent experiments. * P < 0.05. ( D ) Immunoblot analysis of GATA-2 in K562s treated with siRNAs specific to GATA-2 (si_GATA-2) for 48 h or K562s transfected with a construct overexpressing GATA-2 (over_GATA-2) for 48 h, respectively. ( E ) Q-PCR analysis of Pri-27a∼24, mature miR-27a and miR-24 expression in K562s treated as described in (D). ( F ) ChIP-PCR analysis of GATA-1 and GATA-2 occupancy at the −557 site in K562s transfected with control siRNAs or siRNAs specific to GATA-1/GATA-2 or K562s transfected with empty pcDNA3.1 vectors or pcDNA3.1_GATA-1/GATA-2. ( G ) ChIP-PCR and ChIP-q-PCR analysis of GATA-1 and GATA-2 occupancy at the −557 site in CD34+ HPCs of E culture. ( H ) Q-PCR analysis of Pri-27a-24 abundance in CD34+ HPCs of E culture (Top panel). Error bars represent the standard deviation obtained from three independent experiments. An immunoblot analysis of GATA-1 and GATA-2 expression in CD34+ HPCs of E culture (Bottom panel). ( I ) Q-PCR (left panel) and immunoblot (right panel) analysis of GATA-1 and GATA-2 expression in CD34+ HPCs transduced with lentivirus-expressing siRNAs against GATA-1 or control on day 11 of E culture. Error bars represent standard deviation obtained from three independent experiments. * P < 0.05; ** P < 0.01. ( J ) Q-PCR analysis of Pri-27a∼24, mature miR-27a and miR-24 expression in CD34+ HPCs treated as described in (I). Error bars represent the standard deviation obtained from three independent experiments. ** P < 0.01.

Article Snippet: CD34+ cells were enriched from mononuclear cells through positive immunomagnetic selection (CD34 MultiSort kit, Miltenyi Biotec, Bergisch-Glad-bach, Germany).

Techniques: Expressing, Standard Deviation, Western Blot, Functional Assay, Activity Assay, Mutagenesis, Luciferase, Transfection, Construct, Control, Transduction

Journal: Nucleic Acids Research

Article Title: A regulatory circuit comprising GATA1/2 switch and microRNA-27a/24 promotes erythropoiesis

doi: 10.1093/nar/gkt848

Figure Lengend Snippet: A regulatory circuit involving GATA-1, GATA-2 and miR-27a/24 in erythropoiesis. ( A ) A schematic representation of the regulatory circuit comprised GATA-1, GATA-2, miR-27a and miR-24 in erythroid differentiation. ( B , C ) ChIP-PCR and ChIP-q-PCR analysis of GATA-1 and GATA-2 occupancy at the −557 site in K562s transfected with scramble or miRNA mimics and inhibitors (B, r miR-27a and C, miR-24). ( D ) Q-PCR analysis of Pri-27a∼24 abundance in K562s treated as described in (B) and (C). Error bars represent the standard deviation obtained from three independent experiments. * P < 0.05; ** P < 0.01. ( E ) Q-PCR analysis of Pri-27a∼24 abundance in CD34+ HPCs transduced with Lenti-miR-27a/24 or Lenti-GFP control for 11 days. Error bars represent the standard deviation obtained from three independent experiments. * P < 0.05. ( F ) Q-PCR analysis of Pri-27a∼24 abundance in CD34+ HPCs transduced with Zip-miRNA or Zip-GFP for 11 days. Error bars represent the standard deviation obtained from three independent experiments. ** P < 0.01.

Article Snippet: CD34+ cells were enriched from mononuclear cells through positive immunomagnetic selection (CD34 MultiSort kit, Miltenyi Biotec, Bergisch-Glad-bach, Germany).

Techniques: Transfection, Standard Deviation, Transduction, Control

Journal: International journal of biological sciences

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

doi: 10.7150/ijbs.79913

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

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

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

Journal: International journal of biological sciences

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

doi: 10.7150/ijbs.79913

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

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

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

Journal: International journal of biological sciences

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

doi: 10.7150/ijbs.79913

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

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

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

Journal: International journal of biological sciences

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

doi: 10.7150/ijbs.79913

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

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

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

Journal: International journal of biological sciences

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

doi: 10.7150/ijbs.79913

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

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

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

Journal: International journal of biological sciences

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

doi: 10.7150/ijbs.79913

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

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

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

Journal: International journal of biological sciences

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

doi: 10.7150/ijbs.79913

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

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

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