Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Rac1 regulates platelet shedding of CD40L in abdominal sepsis.

doi: 10.1038/labinvest.2014.92

Figure Lengend Snippet: Figure 3 CXCL2 induces Rac1 activity in neutrophils. (a) Neutrophils were isolated by magnetic beads and then 1 106 neutrophils were incubated with CXCL2 (0.3 mg/ml) for 20 min with and without Rac1 inhibitor NSC23766 (N6-[2-[[4-(diethylamino)-1-methylbutyl] amino]-6- methyl-4-pyrimidinyl]-2 methyl-4, 6-quinolinediamine trihydrochloride; 10 mM). Active Rac1 protein was pulled down from neutrophil lysates by using GST-PAK beads. Rac1-GTP was detected by western blot. (b) Band intensities were quantified by densitometry and normalized to total Rac1. Bars represent mean±s.e.m. and n ¼ 4. *Po0.05 vs control and #Po0.05 vs vehicle þ CXCL2.

Article Snippet: About 1 106 neutrophils were preincubated with NSC23766 (10 mM) for 20 min before challenging with 0.3 mg/ml recombinant mouse CXCL2 (R&D Systems) or PBS as a control for 30 min at 37 1C.

Techniques: Activity Assay, Isolation, Magnetic Beads, Incubation, Western Blot, Control

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Rac1 regulates platelet shedding of CD40L in abdominal sepsis.

doi: 10.1038/labinvest.2014.92

Figure Lengend Snippet: Figure 5 Rac1 regulates neutrophil secretion of matrix metalloproteinase-9 (MMP-9). (a) Neutrophils were isolated by magnetic beads. (a) Isolated neutrophils were incubated with CXCL2 (0.3 mg/ml) and then was the level of MMP-9 in permeabilized neutrophils determined by confocal microscopy. (b) Summarized data showing mean fluorescence intensity (MFI) of MMP-9 in neutrophils. Bars represent mean±s.e.m. and n ¼ 4. *Po0.05 vs control and #Po0.05 vs vehicle þ CXCL2.

Article Snippet: About 1 106 neutrophils were preincubated with NSC23766 (10 mM) for 20 min before challenging with 0.3 mg/ml recombinant mouse CXCL2 (R&D Systems) or PBS as a control for 30 min at 37 1C.

Techniques: Isolation, Magnetic Beads, Incubation, Confocal Microscopy, Fluorescence, Control

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Rac1 regulates platelet shedding of CD40L in abdominal sepsis.

doi: 10.1038/labinvest.2014.92

Figure Lengend Snippet: Figure 6 Neutrophil-derived matrix metalloproteinase-9 (MMP-9) regulates platelet shedding of CD40L. Isolated MMP-9 gene-deficient platelets were incubated with proteinase-activated receptor-4 (PAR4) and with supernatants from either wild-type or MMP-9-deficient neutrophils stimulated with CXCL2 for 30 min. Soluble CD40L was determined in the supernatants by enzyme-linked immunosorbent assay. Nonstimulated platelets served as control. Bars represent mean±s.e.m. and n ¼ 4-5. *Po0.05 vs control, #Po0.05 vs control, and ¤Po0.05 vs wild-type neutrophil supernatant.

Article Snippet: About 1 106 neutrophils were preincubated with NSC23766 (10 mM) for 20 min before challenging with 0.3 mg/ml recombinant mouse CXCL2 (R&D Systems) or PBS as a control for 30 min at 37 1C.

Techniques: Derivative Assay, Isolation, Incubation, Enzyme-linked Immunosorbent Assay, Control

Journal: Acta pharmaceutica Sinica. B

Article Title: Pancreatic melatonin enhances anti-tumor immunity in pancreatic adenocarcinoma through regulating tumor-associated neutrophils infiltration and NETosis.

doi: 10.1016/j.apsb.2023.01.020

Figure Lengend Snippet: Figure 5 Neutrophil recruitment was induced by CXCL2 secreted from pancreatic tumors under melatonin stimulation. (A) PKH26-positive neutrophil populations re-supplemented into the mice inside the tumor were determined using flow cytometry. (B, C) Maturation and migration of neutrophils induced by TCDS with melatonin added were studied using flow cytometry and Transwell migration assay. (D, E) Maturation and migration of neutrophils induced by melatonin pre-treated TCDS were studied using flow cytometry and Transwell migration assay. Scale bar: 100 mm. (F) Cytokines and chemokines expressions were determined using a protein array using TCDS with or without melatonin treatment. The expression levels were measured using ImageJ. Cytokines with significant changes (P < 0.05) were marked red, while CXCL2 was marked blue. (G) Expression levels of the cytokines and chemokines were plotted using the melatonin-treated group against the control group. (H) A graph of CXCL2 expression level in human pancreatic cancer tissue. (I) The measurements of CXCL2 level in the control or melatonin group were determined using ELISA. (J) MT1 and MT2 levels from tumor cells, TAMs and TANs were determined by flow cytometry. Tumor cells were isolated from tumor tissues extracted from the mice model and identified as CD45e cells. TAMs were isolated from tumor tissues and identified as CD11bþF4/80þ cells. (K) Relative expression levels of CXCL2 from panc02 tumor cells (left) and TAMs (right) were determined by flow cytometry. (L) CXCL2 mRNA (left) and protein expression (right) levels were determined using qRT-PCR and immunoblotting. (M) CXCL2 mRNA expression level derived from mock and CXCL2-knockdown (KD) Panc02 cells. (N) Neutrophils induced by TCDS derived from mock or KD cells with or without melatonin treatment were co-cultured with wild-type Panc02 cells. Cell apoptosis was determined by Annexin V staining followed by flow cytometry. (O) Neutrophil migration assay was performed on the Transwell system. Numbers of migrated cells within mi- croscope are summarized in the graph Scale bar: 200 mm. (n Z 3). Data are shown as mean SD. *P < 0.05; **P < 0.01; ***P < 0.001. Abbreviations: NT, normal tissue; T, tumor tissue; TCDS, tumor cell-derived supernatant; Veh, Vehicle-treated TCDS.

Article Snippet: Antibodies against CXCL2 (MAB452) was purchased from R&D Systems (USA) and b-actin (#4970) from Cell Signaling Technology.

Techniques: Cytometry, Migration, Transwell Migration Assay, Protein Array, Expressing, Control, Enzyme-linked Immunosorbent Assay, Isolation, Quantitative RT-PCR, Western Blot, Derivative Assay, Knockdown, Cell Culture, Staining

Journal: Acta pharmaceutica Sinica. B

Article Title: Pancreatic melatonin enhances anti-tumor immunity in pancreatic adenocarcinoma through regulating tumor-associated neutrophils infiltration and NETosis.

doi: 10.1016/j.apsb.2023.01.020

Figure Lengend Snippet: Figure 7 Clinical significance of melatonin-induced CXCL2 expression and NETosis in human PAAD patients. (A) Representative images of a tissue section on the human pancreatic tissue microarray. Scale bar, 20 mm. (BeD) Linear regression analysis of the fluorescence signal intensities from the human PAAD tissue microarray. The correlation of melatonin against CXCL2, melatonin against MPO, and CXCL2 against MPO were analyzed. P values are shown on the graphs. (E) Linear regression analysis of fluorescence signal intensities from the human PAAD tissue microarray. The melatonin levels were plotted against the H3Cit-positive neutrophils. P value is shown on the graph. (FeI) Progress-free survival plot of PAAD patients using FSI of CXCL2 with melatonin, MPO with melatonin, CXCL2 with MPO, and H3Cit with melatonin expressions as cross-correlation factors. Data are shown as mean SD. *P < 0.05; ***P < 0.001; ****P < 0.0001.

Article Snippet: Antibodies against CXCL2 (MAB452) was purchased from R&D Systems (USA) and b-actin (#4970) from Cell Signaling Technology.

Techniques: Expressing, Microarray

Journal: Acta pharmaceutica Sinica. B

Article Title: Pancreatic melatonin enhances anti-tumor immunity in pancreatic adenocarcinoma through regulating tumor-associated neutrophils infiltration and NETosis.

doi: 10.1016/j.apsb.2023.01.020

Figure Lengend Snippet: Figure 8 Schematic diagram of the mechanism underlying the anti- tumor effect of melatonin on PAAD. Supplementation of melatonin, in which production is suppressed in patients, represses PAAD pro- gression in the animal model. Melatonin treatment induced CXCL2 production in the pancreatic tumor tissues, attracting TANs infiltra- tion. The attracted neutrophils present with anti-tumor N1-like phe- notypes, and promote anti-tumor effects by the action of NETs. This observed mechanism is confirmed in clinical samples.

Article Snippet: Antibodies against CXCL2 (MAB452) was purchased from R&D Systems (USA) and b-actin (#4970) from Cell Signaling Technology.

Techniques: Animal Model