Journal: Redox Biology

Article Title: Development of KEAP1-targeting PROTAC and its antioxidant properties: In vitro and in vivo

doi: 10.1016/j.redox.2023.102783

Figure Lengend Snippet: SD2267 attenuated acetaminophen (APAP)-induced liver injury in mice (A) Experimental schedule of the APAP-induced liver injury mouse model. After 16 h of fasting, mice were injected with APAP (250 mg/kg, IP) and 2 h later treated with SD2267 (1 or 3 mg/kg, IP) for another 6 h. (B) Serum AST and ALT levels are presented as means ± SEMs. (C) Protein expression levels of liver tissues were determined by western blotting, and densitometry results of each protein levels are presented as means ± SEMs (n = 3 per group). (D) GSH to GSSG ratio was detected from liver tissues, and presented as means ± SEMs (n = 3 per group). (E) Representative images of hematoxylin and eosin (HE) stained liver tissues (left) with measured areas of necrosis (presented as means ± SEMs) (right). Necrotic areas are marked with dashed lines in HE images. (F) Representative images of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) stained liver tissues (left) and the number of TUNEL positive cells per high power field (HPF; x400) (presented as means ± SEMs) (right). cv; central vein. Scale bar = 100 μm * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Sections were stained with hematoxylin and eosin (HE) staining or terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining using a TUNEL assay kit (Elabscience, #E-CK-A331, Wuhan, China).

Techniques: Injection, Expressing, Western Blot, Staining, TUNEL Assay

Journal: Liver Transplantation

Article Title: The Impact of Dabigatran Treatment on Sinusoidal Protection Against Hepatic Ischemia/Reperfusion Injury in Mice

doi: 10.1002/lt.25929

Figure Lengend Snippet: Characterization of thrombin generation after hepatic IRI and cytotoxicity of thrombin for hepatic SECs. (A) In the clinical study of patents who underwent hepatectomy (n = 14), plasma TAT levels were plotted at 2 time points: before and 1 day after hepatectomy. TAT levels were significantly elevated after operation. (B) In in vivo hepatic IRI models of mice, plasma TAT levels measured by ELISA was significantly increased after IRI compared with the naïve group (n = 6 in each group). (C‐a) In hepatic SECs cultured in the nonischemic condition, LDH cytotoxicity levels in the supernatant of cell cultures were elevated in a dose‐dependent manner with the addition of thrombin. The addition of 100 U/mL thrombin significantly increased LDH cytotoxicity compared with both groups of naïve and 10 U/mL thrombin (n = 4 in each group). (C‐b) In H‐R models of SECs in vitro, the addition of 100 U/mL thrombin also significantly increased LDH cytotoxicity compared with vehicle (n = 5 in each group). H‐R was established using an anaeropack jar system.

Article Snippet: Cell cytotoxicity was assessed by measuring lactate dehydrogenase (LDH) levels in the supernatant using a Cytotoxicity LDH Assay Kit‐WST (Dojindo, Japan) following the manufacturer’s instructions.

Techniques: Clinical Proteomics, In Vivo, Enzyme-linked Immunosorbent Assay, Cell Culture, In Vitro

Journal: Liver Transplantation

Article Title: The Impact of Dabigatran Treatment on Sinusoidal Protection Against Hepatic Ischemia/Reperfusion Injury in Mice

doi: 10.1002/lt.25929

Figure Lengend Snippet: Direct effect of dabigatran treatment on pure cultured hepatic SECs in an H‐R model in vitro. (A) In H‐R models of pure cultured hepatic SECs, dabigatran treatment significantly decreased LDH cytotoxicity levels in the supernatant compared with vehicle (n = 5 in each group). (B‐a) Dabigatran treatment significantly increased TM expression in cell lysate measured by ELISA compared with vehicle (n = 5 in each group). (B‐b) By contrast, in TM expression in the supernatant of cell cultures measured by ELISA, there was no significant difference between the vehicle and dabigatran groups (n = 5 in each group). (C‐a) Based on Western blot analysis, dabigatran treatment did not significantly affect HMGB‐1 expression in cell lysate compared with the vehicle group (n = 5 in each group). Quantification of HMGB‐1 band intensities normalized to β‐actin. (C‐b) By contrast, HMGB‐1 levels in the supernatant of cell cultures measured by ELISA were markedly reduced by dabigatran treatment compared with the vehicle (n = 5 in each group). In H‐R models of this study, SECs were cultured in a serum‐starved medium containing 100 U/mL thrombin and exposed to H‐R using an anaeropack jar system.

Article Snippet: Cell cytotoxicity was assessed by measuring lactate dehydrogenase (LDH) levels in the supernatant using a Cytotoxicity LDH Assay Kit‐WST (Dojindo, Japan) following the manufacturer’s instructions.

Techniques: Cell Culture, In Vitro, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Liver Transplantation

Article Title: The Impact of Dabigatran Treatment on Sinusoidal Protection Against Hepatic Ischemia/Reperfusion Injury in Mice

doi: 10.1002/lt.25929

Figure Lengend Snippet: Direct effect of dabigatran treatment on pure cultured hepatocytes in an H‐R model in vitro. In H‐R models of pure cultured hepatocytes, there was no significant difference between the H‐R + vehicle group and the H‐R + dabigatran group (A) in LDH cytotoxicity levels in the supernatant of cell cultures, in TM levels in cell lysate measured by ELISA (B‐a), in TM levels in the supernatant of cell cultures measured by ELISA (B‐b), in HMGB‐1 expression in cell lysate evaluated by Western blot analysis (C‐a), or in HMGB‐1 levels in the supernatant of cell cultures measured by ELISA (C‐b) (n = 5 in each group). In Western blot analysis, quantification of HMGB‐1 band intensities normalized to β‐actin. In H‐R models of this study, cells were cultured in a serum‐starved medium containing 100 U/mL thrombin and exposed to H‐R using an anaeropack jar system.

Article Snippet: Cell cytotoxicity was assessed by measuring lactate dehydrogenase (LDH) levels in the supernatant using a Cytotoxicity LDH Assay Kit‐WST (Dojindo, Japan) following the manufacturer’s instructions.

Techniques: Cell Culture, In Vitro, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot

Journal: Liver Transplantation

Article Title: The Impact of Dabigatran Treatment on Sinusoidal Protection Against Hepatic Ischemia/Reperfusion Injury in Mice

doi: 10.1002/lt.25929

Figure Lengend Snippet: Effects of dabigatran for hepatocytes in a paracrine communication model between hepatic SECs and hepatocytes. (A) A scheme of our paracrine communication model between hepatic SECs and hepatocytes. SECs were pretreated with or without dabigatran followed by incubation in a serum‐starved medium in the H‐R condition; thereafter the supernatant of the SECs was harvested. Hepatocytes were pretreated with the supernatant of SECs followed by incubation in a serum‐starved medium in the H‐R condition. (B) LDH cytotoxicity levels in the supernatant of hepatocyte cultures were significantly lower in the group of H‐R + the supernatant of SECs pretreated with dabigatran than in the group of H‐R + the supernatant of SECs pretreated with vehicle (n = 5 in each group). TUNEL staining detected cellular death of hepatocytes after H‐R (C‐a) in the group of H‐R + the supernatant of SECs pretreated with vehicle and (C‐b) in the group of H‐R + the supernatant of SECs pretreated with dabigatran (original magnification ×200). (C‐c) The supernatant of SECs pretreated with dabigatran markedly reduced the number of TUNEL‐positive hepatocytes compared with the supernatant of SECs pretreated with the vehicle (n = 5 in each group).

Article Snippet: Cell cytotoxicity was assessed by measuring lactate dehydrogenase (LDH) levels in the supernatant using a Cytotoxicity LDH Assay Kit‐WST (Dojindo, Japan) following the manufacturer’s instructions.

Techniques: Incubation, TUNEL Assay, Staining

Journal: Gastroenterology

Article Title: Cysteine-rich domains of muc3 intestinal mucin promote cell migration, inhibit apoptosis, and accelerate wound healing.

doi: 10.1053/j.gastro.2006.09.006

Figure Lengend Snippet: Figure 6. Effect of Muc3 and MUC3A expression on apoptosis. (A) Western blotting using anti-Flag antibody. Lanes: stably transfected LoVo clone LhM3c14 cytoplasmic fraction (LhM3c14 cyt) and membrane fraction (LHM3C14 MEM), mock-transfected LoVo cell clone membrane fraction (LMOCK MEM), nontransfected LoVo membrane fraction (MEM). (B) Percentage change in apoptosis with () or without () TNF- (100 ng/mL) treatment for 48 hours. Cell lines included parental LoVo, LhM3c14, Lmock, and nontransfected LoVo cells pretreated with m3EGF1,2 (10 g/mL) or GST (5 g/mL) for 1 hour before addition of TNF-. Baseline apoptosis 3.9%. *P .016 vs LoVo (), ^P .009 vs GST, P .002 vs Lmock; n 2–3 plates/treatment and 3 fields counted/plate. (C) Percentage change in apoptosis in nontransfected LoVo cells with () or without () sequential interferon- and anti-Fas antibody treatment for 72 hours. Cells were pretreated with m3EGF1,2 (10 g/mL), m3EGF1 (10 g/mL), m3EGF2 (10 g/mL), GST (10 g/mL), EGF (10 ng/mL), or m3EGF1 (5 g/mL) m3EGF2 (5 g/mL) before additionofanti-Fasantibody.Baselineapoptosis1.1%and.9%forleftandrightpanels,respectively(ANOVA,P.001,withinbothpanels).*P.005vsLoVo, LoVo m3EGF1, m3EGF2 and GST controls (left panel). *P .004 vs LoVo and LoVo m3EGF1 m3EGF2 (right panel); n 2 plates/treatment and 2 fields counted/plate. (D) Percentage change in apoptosis with () or without () sequential interferon- and anti-Fas antibody treatment for 48 hours. Cell lines included LhM3c14andLmock(ANOVA,P.001).Baselineapoptosis2.3%.*P.025vsLmock()andLhMc14()n3plates/treatmentand3fieldscounted/plate. (E) LoVo cells were treated with increasing concentrations of the EGF-receptor inhibitor, tyrphostin (AG1478). This resulted in a dose-dependent reversal of the anti-apoptosis effect of recombinant EGF, but did not affect the anti-apoptosis effect of m3EGF1,2 (ANOVA, P .001). Baseline apoptosis 2.0%. *P .03 vs m3EGF1,2 (same tyrphostin concentrations). (Note that maximal apoptosis observed varied from experiment to experiment because of the limited half-life of the activity of the anti-Fas in storage).

Article Snippet: Apoptosis was measured in histologic specimens using a erminal deoxynucleotidyl transferase–mediated deoxyuridine riphosphate biotin nick-end labeling (TUNEL) assay (TACS.XL n Situ Apoptosis Detection Kit; R&D Systems), according to he manufacturer’s directions.

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

Journal: Gastroenterology

Article Title: Cysteine-rich domains of muc3 intestinal mucin promote cell migration, inhibit apoptosis, and accelerate wound healing.

doi: 10.1053/j.gastro.2006.09.006

Figure Lengend Snippet: Figure 9. Effect of recombinant Muc3 protein on acetic acid–in- duced apoptosis. (A) Mean number of apoptotic cells determined by TUNEL assay in the distal colon of mice 30 hours after acetic acid administration in mice treated twice with 100 g m3EGF1,2 or control peptide 100 g BSA in PBS (n 10 mice each treatment). (B) Repre- sentative distal colon 30 hours after acetic acid and control enema treatments. TUNEL-positive nuclei are stained darkly. (C) Representa- tive distal colon 30 hours after acetic acid and m3EGF1,2 enema treat- ments (magnification, 200).

Article Snippet: Apoptosis was measured in histologic specimens using a erminal deoxynucleotidyl transferase–mediated deoxyuridine riphosphate biotin nick-end labeling (TUNEL) assay (TACS.XL n Situ Apoptosis Detection Kit; R&D Systems), according to he manufacturer’s directions.

Techniques: Recombinant, TUNEL Assay, Control, 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 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: Molecules

Article Title: Semi-Synthetic Ingenol Derivative from Euphorbia tirucalli Inhibits Protein Kinase C Isotypes and Promotes Autophagy and S-Phase Arrest on Glioma Cell Lines

doi: 10.3390/molecules24234265

Figure Lengend Snippet: Effect of IngC on apoptosis pathway on glioma cell lines. ( A,B ) Panel of 35 proteins related to apoptosis. The data represented by the heat maps show the proteins modulated after 24 and 72 h of IngC treatment (3X IC 50 value (13.09 μM) in glioma cells, GAMG and U373. ( C ) Cells were treated with 3X IC 50 concentrations of IngC (24 and 72 h) for the indicated time periods. GAMG and U373 cell lysates (20 μg per lane) were analyzed using immunoblotting with anti-DR5. The tubulin was used as an internal control to normalize the amount of proteins applied in each lane. This data is representative of three independent experiments. ( D ) After the IngC treatment with 3X IC 50 for 72 h, U373 cells were fixed, stained with annexin V-FITC/PI and analyzed by FACScan. Data shown are representative of three independent experiments. ( E ) After IngC treatment with 3X IC 50 for 72 h, DNA fragmentation in U373 cell line was measured with the TUNEL assay using flow cytometry. The graphs are representative of at least three independent experiments performed in duplicate. n.s. means non-significant. ** p < 0.005

Article Snippet: The relative protein expression levels of a panel of 35 proteins related to apoptosis and 26 proteins related to cellular stress were obtained while using the Proteome Profiler Human Apoptosis Array (R&Dsystems- #ARY009) and Proteome Profiler Human Cell Stress Array (R&Dsystems-#ARY018), according to the manufacturer’s instructions and as previously reported [ ].

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

Journal: Molecules

Article Title: Semi-Synthetic Ingenol Derivative from Euphorbia tirucalli Inhibits Protein Kinase C Isotypes and Promotes Autophagy and S-Phase Arrest on Glioma Cell Lines

doi: 10.3390/molecules24234265

Figure Lengend Snippet: IngC promotes autophagy on glioma cells. ( A ) Cells were treated with the IC 50 value of IngC for the indicated time periods. GAMG cell lysates (20 μg per lane) were analyzed using immunoblotting with anti-LC3. ( A , B ) are representative of three independent experiments with GAMG. Tubulin was used as an internal control to normalize the amount of proteins applied in the treatment without bafilomycin A1 (Baf). ( C ) Development of AVO in IngC-treated cells by detecting green and red fluorescence in acridine orange-stained cells using FACS analysis. U373 cells were treated with IngC (IC 50 value), and Baf (20 nM) for 72 h. The graphs are representative of at least two independent experiments. FITC indicates green color intensity, while PerCP shows red color intensity. ( D ) Effect of baf on GAMG and U373 cell viability of IngC-treated cells. At 3 h after exposure to IngC, baf was added and cultured until 72 h and evaluated by MTS assay. The viability of the untreated cells was considered as 100%. Results shown are the means ± S.D. of three independent experiments. ( E ) Effect of Baf on IngC-induced apoptosis. After, IngC and bafilomicyn treatment for 72 h, GAMG cells were stained with annexin V-FITC/PI and analyzed by FACScan. Data shown are representative of three independent experiments. ** p < 0.005 and *** p < 0.0001

Article Snippet: The relative protein expression levels of a panel of 35 proteins related to apoptosis and 26 proteins related to cellular stress were obtained while using the Proteome Profiler Human Apoptosis Array (R&Dsystems- #ARY009) and Proteome Profiler Human Cell Stress Array (R&Dsystems-#ARY018), according to the manufacturer’s instructions and as previously reported [ ].

Techniques: Western Blot, Fluorescence, Staining, Cell Culture, MTS Assay