Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Oral NaHCO 3 activates a splenic anti-inflammatory pathway; evidence cholinergic signals are transmitted via mesothelial cells

doi: 10.4049/jimmunol.1701605

Figure Lengend Snippet: Antibodies and Reagents utilized

Article Snippet: F4/80-A488, EMRI Bioss bs-7058R-A488 AE084243 100ul rabbit http://biossusa.com/store/datasheets/bs-7058R-A488 IL-10-Alexa647, CC320 Novus NB100-63026AF6477 1007-081415 250ul mouse ab http://www.novusbio.com/IL-10-Antibody-CC320_NB100-63026AF647.html CD44-FITC, OX-49 BD Bioscience 550974 4324539 .2ml mouse α rat Zimmerman MA, et. al ., Am J Renal Physiology 2015( 29 )& Tipton Am J PhysiolRegulIntegr Comp Physiol 2012( 28 ) CD69-PE, H1.2F3 eBioscience 12-0691-82 E01332-1634 .5ml α-mouse Yokoyama W, et. al ., JImmunol, 1988, 141( 2 ): 369-76(64).

Techniques: Cell Differentiation

Journal: Journal for Immunotherapy of Cancer

Article Title: Generation of non-genetically modified, CAR-like, NK cells

doi: 10.1136/jitc-2024-009070

Figure Lengend Snippet: eNK cells can efficiently be armed with multiple Pin-monoclonal antibodies and remain actively cytotoxic. ( A, B and C ) eNK cells were armed with Pin-CD20, Pin-CD19 or both (double-armed eNK) using fluorescently labeled pin-CD20 (Pin-CD20-AF647) and pin-CD19 (Pin-CD19-AF488) and analyzed 1 hour, 8 hours, 24 hours, 48 hours and 72 hours after. ( A ) Representative dot plots of Pin-CD20-AF647 and Pin-CD19-AF488 arming for each condition. ( B ) Frequency of eNK cells armed with Pin-CD20 (left) or Pin-CD19 (right). ( C ) Frequency of eNK cells armed simultaneously with both Pin-CD20 and Pin-CD19 (double-armed). n=4 eNK donors. Data are shown as mean±SEM. ( D ) Patient with lymphoma cells were co-cultured with eNK or Pin-CD20 and/or Pin-CD19 eNK for 16 hours. Tumor cell death was assessed by flow cytometry. Mean±SEM is shown, MCL, n=3; DLBCL, n=4; FL, n=3. For each patient sample, two to three eNK donors were tested. ( E ) 1×10 6 eNK, Pin-CD20 eNK, Pin-CD19 eNK or double-armed eNK were intraperitoneally co-injected with 1×10 6 Daudi and 1×10 6 Nalm6 cells into NSG mice. Peritoneal lavage were performed after 4 hours and the number of target cell was analyzed by flow cytometry. Vehicle, n=3 mice; eNK, n=5 mice; Pin-CD20 eNK, n=5 mice; Pin-CD19 eNK, n=5 mice; double-armed eNK, n=5 mice. Two-way analysis of variance with Sidak’s test and mean±SEM are shown, *p≤0.05; **p≤0.01; ***p≤0.001; ****p≤0.0001; ns, not significant. eNK, expanded natural killer; DLBCL, diffuse large B cell lymphoma; FC, follicular lymphoma; MCL, mantle cell lymphoma.

Article Snippet: For Pin-mAbs detection staining, either anti-idiotype anti-rituximab-AF488 or AF647 (RnD systems, FAB9630G and FAB9630R) or anti-human-Fab-AF647 (CliniScience, CSA3835) were used.

Techniques: Labeling, Cell Culture, Flow Cytometry, Injection

Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: Deficiency of mouse mast cell protease 4 mitigates cardiac dysfunctions in mice after myocardium infarction

doi: 10.1016/j.bbadis.2019.01.011

Figure Lengend Snippet: Chymase mMCP4 expression and function in mice after MI injury. A. Western blot analysis of mouse chymase mMCP4 expression in sham-operated and 28 days post-MI hearts from WT mice. B. Immunofluorescent double staining colocalized mMCP4-positive cells to myosin heavy chain-positive cardiomyocytes in the infarct, border, and remote regions in WT mice at 28 days post-MI. C. Immunofluorescent double staining showed mMCP4 expression in α-SMA-positive fibroblasts and Mac-2-positive macrophages in the infarct regions from 28 days post-MI heart. Scale: 200 μm, inset scale: 70 μm. D. Mortality rate of both WT and Mcpt4−/− mice during the course of 28 days of post-MI recovery. Kaplan-Meier survival analysis with log-rank test. E. Infarct sizes of both WT and Mcpt4−/− mice at 3 days and 28 days post-MI. Representative M-mode echocardiography images (F), ejection fraction (EF) (G), fractional shortening (FS) (H), body weight, heart weight, and heart-to-body weight ratio (I), LV diastole and systole volumes (J), and LV diastole and systole internal diameters (K) of WT and Mcpt4−/− mice at 28 days after sham operation or MI. The mouse numbers and genotypes are indicated in the legends. P<0.05 was considered statistically significant, independent t-test or one-way ANOVA with post-hoc Bonferroni test. Data are mean±SEM.

Article Snippet: Immunofluorescent, immunohistological, and Masson’s trichrome staining To localize apoptotic cells and detect the expression of mMCP4 in the heart, heart frozen sections (6 μm) were used for immunofluorescent staining for cardiomyocyte (myosin heavy chain, 1:50, Cat# bs-15444R-A488, Bioss In.

Techniques: Expressing, Western Blot, Double Staining

Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: Deficiency of mouse mast cell protease 4 mitigates cardiac dysfunctions in mice after myocardium infarction

doi: 10.1016/j.bbadis.2019.01.011

Figure Lengend Snippet: Cell apoptosis in post-MI myocardium. A. Immunofluorescent double staining colocalized cleaved caspase 3-positive cells to myosin heavy chain-positive cardiomyocytes in infarct, border, and remote regions from WT (top panels) and Mcpt4−/− mice (bottom panels) at 28 days post-MI. B. Quantification of apoptotic cardiomyocytes in infarct, border, and remote regions. C. Immunofluorescent double staining colocalized cleaved caspase 3-positive cells to α-SMA-positive fibroblasts in infarct, border, and remote regions from WT (top panels) and Mcpt4−/− mice (bottom panels) at 28 days post-MI. D. Quantification of fibroblast apoptosis in infarct, border, and remote regions. Scale: 200 μm, inset scale: 70 μm. Data are representative of 6~8 specimens per genotype. P<0.05 was considered statistically significant, independent t-test. Data are mean±SEM.

Article Snippet: Immunofluorescent, immunohistological, and Masson’s trichrome staining To localize apoptotic cells and detect the expression of mMCP4 in the heart, heart frozen sections (6 μm) were used for immunofluorescent staining for cardiomyocyte (myosin heavy chain, 1:50, Cat# bs-15444R-A488, Bioss In.

Techniques: Double Staining

Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: Deficiency of mouse mast cell protease 4 mitigates cardiac dysfunctions in mice after myocardium infarction

doi: 10.1016/j.bbadis.2019.01.011

Figure Lengend Snippet: Role of mMCP4 in cardiomyocyte apoptosis. A/B. Representative FACS analysis and quantification of Annexin V+PI− early apoptotic cardiomyocytes and Annexin V+PI+ late apoptotic cardiomyocytes from WT and Mcpt4−/− mice after cells were treated with and without (Control) H2O2. C/D. Annexin V and PI immunofluorescent staining representative images and apoptosis quantification of cardiomyocytes from WT and Mcpt4−/− mice with and without 100 μM H2O2 treatment. Data are mean±SEM from six independent experiments. Scale: 200 μm, inset scale: 70 μm. E. Immunoblot analysis of Bax, Bid, and tBid in cardiomyocytes from WT and Mcpt4−/− mice after cells were treated with or without H2O2. F. Immunoblot analysis of CatS, CatK, CatL, and CatB in cardiomyocytes from WT and Mcpt4−/− mice after cells were treated with or without H2O2. The same blots were reprobed for β-Actin to ensure equal protein loading. Immunoblot data are representative of three independent experiments. P<0.05 was considered statistically significant, independent t-test or one-way ANOVA with post-hoc Bonferroni test. Data are mean±SEM.

Article Snippet: Immunofluorescent, immunohistological, and Masson’s trichrome staining To localize apoptotic cells and detect the expression of mMCP4 in the heart, heart frozen sections (6 μm) were used for immunofluorescent staining for cardiomyocyte (myosin heavy chain, 1:50, Cat# bs-15444R-A488, Bioss In.

Techniques: Staining, Western Blot

Journal: International Journal of Biological Sciences

Article Title: SUMOylation of Nuclear γ-Actin by SUMO2 supports DNA Damage Repair against Myocardial Ischemia-Reperfusion Injury

doi: 10.7150/ijbs.74407

Figure Lengend Snippet: Intracellular localization of γ-actin and DNA damage in a mouse model of myocardial infarction. (A) Immunofluorescence imaging of intracellular γ-actin. Following the induction of myocardial infarction by LAD ligation, cardiac tissues from C57BL/6N mice were analyzed by immunofluorescence imaging with anti-γ-actin antibody. Scale bar: 20 µm. (B) Western blot nalysis of cytosolic and nuclearγ-actin. Following the induction of myocardial infarction by LAD ligation, cytosolic and nuclear proteins were isolated from the cardiac tissues and analyzed by Western blotting with anti-γ-actin antibody. The blots were quantified by a densitometric method. (C) TUNEL staining of cardiac tissues. Following the induction of myocardial infarction by LAD ligation, cardiac tissues were stained with TUNEL staining. The cell nuclei were stained with DAPI. The images were quantified by ImageJ software. Scale bar: 50 µm. (D) Immunostaining of 8-OHdG. Following the induction of myocardial infarction by LAD ligation, cardiac tissues were probed with anti-8-OHdG antibody and visualised with Alexa Fluor 647-conjugated secondary antibody. The cell nuclei were stained with DAPI. The images were quantified by ImageJ software. Scale bar: 50 µm. (E) Western blot analysis of p-γ-H2ax. Following the induction of myocardial infarction by LAD ligation, cardiac tissues were analyzed by Western blotting with anti-p-γ-H2ax antibody. The blots were quantified by a densitometric method. The results were shown as mean ± SD (n = 6), * p < 0.05.

Article Snippet: The bound antibodies were detected with Alexa Fluor 488/647 anti-mouse IgG secondary antibody at room temperature for 2 h. After three washes with PBS, the cell nuclei were stained with DAPI for 5 min. Immunofluorescence co-localization analysis was performed on LSM 980 laser scanning confocal microscope from Carl Zeiss (Jena, Germany) according to the previous publication .

Techniques: Immunofluorescence, Imaging, Ligation, Western Blot, Isolation, TUNEL Assay, Staining, Software, Immunostaining

Journal: International Journal of Biological Sciences

Article Title: SUMOylation of Nuclear γ-Actin by SUMO2 supports DNA Damage Repair against Myocardial Ischemia-Reperfusion Injury

doi: 10.7150/ijbs.74407

Figure Lengend Snippet: Intracellular localization of γ-actin and DNA damage in H9c2 cell model. (A) Immunofluorescence imaging of intracellular γ-actin. After exposure to 3 h hypoxia (0.1% O 2 ) and 24 h reoxygenation (H/R), H9c2 cells were analyzed by immunofluorescence imaging with anti-γ-actin antibody. Scale bar: 50 µm. (B) Western blot nalysis of cytosolic and nuclear proteins. Following H/R treatment, cytosolic and nuclear proteins were isolated from H9c2 cells and analyzed by Western blotting with anti-γ-actin antibody. The blots were quantified by a densitometric method. (C) TUNEL staining of H9c2 cells. Following H/R treatment, H9c2 cells were stained with TUNEL staining. The cell nuclei were stained with DAPI. The images were quantified by ImageJ software. Scale bar: 100 µm. (D) Immunostaining of 8-OHdG. Following H/R treatment, H9c2 cells were probed with anti-8-OHdG antibody and visualised with Alexa Fluor 647-conjugated secondary antibody. The cell nuclei were stained with DAPI. The images were quantified by ImageJ software. Scale bar: 50 µm. (E) Western blot analysis of p-γ-H2ax. Following H/R treatment, H9c2 cells were analyzed by Western blotting with anti-p-γ-H2ax antibody. The blots were quantified by a densitometric method. The results were shown as mean ± SD (n = 3), * p < 0.05.

Article Snippet: The bound antibodies were detected with Alexa Fluor 488/647 anti-mouse IgG secondary antibody at room temperature for 2 h. After three washes with PBS, the cell nuclei were stained with DAPI for 5 min. Immunofluorescence co-localization analysis was performed on LSM 980 laser scanning confocal microscope from Carl Zeiss (Jena, Germany) according to the previous publication .

Techniques: Immunofluorescence, Imaging, Western Blot, Isolation, TUNEL Assay, Staining, Software, Immunostaining

Journal: International Journal of Biological Sciences

Article Title: SUMOylation of Nuclear γ-Actin by SUMO2 supports DNA Damage Repair against Myocardial Ischemia-Reperfusion Injury

doi: 10.7150/ijbs.74407

Figure Lengend Snippet: Effects of SUMO silencing on the nuclear translocation of γ-actin and DNA damage in H9c2 cell model. SUMO2 was silenced by introducing lentiviral SUMO2 shRNA into H9c2 cells while the lentiviral vector was used as the control. (A) Immunofluorescence imaging of intracellular γ-actin. After exposure to 3 h hypoxia (0.1% O 2 ) and 24 h reoxygenation (H/R), the transfected H9c2 cells were analyzed by immunofluorescence imaging with anti-γ-actin antibody. Scale bar: 50 µm. (B) Western blot nalysis of cytosolic and nuclear proteins. Following H/R treatment, cytosolic and nuclear proteins were isolated from the transfected H9c2 cells and analyzed by Western blotting with anti-γ-actin antibody. The blots were quantified by a densitometric method. (C) TUNEL staining of the transfected H9c2 cells. Following H/R treatment, H9c2 cells were stained with TUNEL staining. The cell nuclei were stained with DAPI. The images were quantified by ImageJ software. Scale bar: 100 µm. (D) Immunostaining of 8-OHdG. Following H/R treatment, the transfected H9c2 cells were probed with anti-8-OHdG antibody and visualised with Alexa Fluor 647-conjugated secondary antibody. The cell nuclei were stained with DAPI. The images were quantified by ImageJ software. Scale bar: 100 µm. (E) Western blot analysis of p-γ-H2ax. Following H/R treatment, the transfected H9c2 cells were analyzed by Western blotting with anti-p-γ-H2ax antibody. The blots were quantified by a densitometric method. The results were shown as mean ± SD (n = 3), * p < 0.05.

Article Snippet: The bound antibodies were detected with Alexa Fluor 488/647 anti-mouse IgG secondary antibody at room temperature for 2 h. After three washes with PBS, the cell nuclei were stained with DAPI for 5 min. Immunofluorescence co-localization analysis was performed on LSM 980 laser scanning confocal microscope from Carl Zeiss (Jena, Germany) according to the previous publication .

Techniques: Translocation Assay, shRNA, Plasmid Preparation, Control, Immunofluorescence, Imaging, Transfection, Western Blot, Isolation, TUNEL Assay, Staining, Software, Immunostaining

Journal: International Journal of Biological Sciences

Article Title: SUMOylation of Nuclear γ-Actin by SUMO2 supports DNA Damage Repair against Myocardial Ischemia-Reperfusion Injury

doi: 10.7150/ijbs.74407

Figure Lengend Snippet: Effects of SUMO overexpression on the nuclear translocation of γ-actin and DNA damage in H9c2 cell model. SUMO2 was silenced by introducing lentiviral SUMO2 cDNA into H9c2 cells while the lentiviral vector was used as the control. (A) Immunofluorescence imaging of intracellular γ-actin. After exposure to 3 h hypoxia (0.1% O 2 ) and 24 h reoxygenation (H/R), the transfected H9c2 cells were analyzed by immunofluorescence imaging with anti-γ-actin antibody. Scale bar: 50 µm. (B) Western blot nalysis of cytosolic and nuclear proteins. Following H/R treatment, cytosolic and nuclear proteins were isolated from the transfected H9c2 cells and analyzed by Western blotting with anti-γ-actin antibody. The blots were quantified by a densitometric method. (C) TUNEL staining of the transfected H9c2 cells. Following H/R treatment, H9c2 cells were stained with TUNEL staining. The cell nuclei were stained with DAPI. The images were quantified by ImageJ software. Scale bar: 100 µm. (D) Immunostaining of 8-OHdG. Following H/R treatment, the transfected H9c2 cells were probed with anti-8-OHdG antibody and visualised with Alexa Fluor 647-conjugated secondary antibody. The cell nuclei were stained with DAPI. The images were quantified by ImageJ software. Scale bar: 100 µm. (E) Western blot analysis of p-γ-H2ax. Following H/R treatment, the transfected H9c2 cells were analyzed by Western blotting with anti-p-γ-H2ax antibody. The blots were quantified by a densitometric method. The results were shown as mean ± SD (n = 3), * p < 0.05.

Article Snippet: The bound antibodies were detected with Alexa Fluor 488/647 anti-mouse IgG secondary antibody at room temperature for 2 h. After three washes with PBS, the cell nuclei were stained with DAPI for 5 min. Immunofluorescence co-localization analysis was performed on LSM 980 laser scanning confocal microscope from Carl Zeiss (Jena, Germany) according to the previous publication .

Techniques: Over Expression, Translocation Assay, Plasmid Preparation, Control, Immunofluorescence, Imaging, Transfection, Western Blot, Isolation, TUNEL Assay, Staining, Software, Immunostaining

Journal: International Journal of Biological Sciences

Article Title: SUMOylation of Nuclear γ-Actin by SUMO2 supports DNA Damage Repair against Myocardial Ischemia-Reperfusion Injury

doi: 10.7150/ijbs.74407

Figure Lengend Snippet: Effects of SUMOylation inhibitor ML-792 on the nuclear translocation of γ-actin and DNA damage in H9c2 cell model. H9c2 cells were pre-treated with ML-792 or vehicle and subsequently challenged by H/R treatment. the lentiviral vector was used as the control. (A) Immunofluorescence imaging of intracellular γ-actin. H9c2 cells were analyzed by immunofluorescence imaging with anti-γ-actin antibody. Scale bar: 50 µm. (B) Western blot analysis of cytosolic and nuclear proteins. Cytosolic and nuclear proteins were isolated from H9c2 cells and analyzed by Western blotting with anti-γ-actin antibody. The blots were quantified by a densitometric method. (C) TUNEL staining of H9c2 cells. H9c2 cells were stained with TUNEL staining. The cell nuclei were stained with DAPI. The images were quantified by ImageJ software. Scale bar: 100 µm. (D) Immunostaining of 8-OHdG. H9c2 cells were probed with anti-8-OHdG antibody and visualised with Alexa Fluor 647-conjugated secondary antibody. The cell nuclei were stained with DAPI. The images were quantified by ImageJ software. Scale bar: 50 µm. (E) Western blot analysis of p-γ-H2ax. H9c2 cells were analyzed by Western blotting with anti-p-γ-H2ax antibody. The blots were quantified by a densitometric method. The results were shown as mean ± SD (n = 3), * p < 0.05.

Article Snippet: The bound antibodies were detected with Alexa Fluor 488/647 anti-mouse IgG secondary antibody at room temperature for 2 h. After three washes with PBS, the cell nuclei were stained with DAPI for 5 min. Immunofluorescence co-localization analysis was performed on LSM 980 laser scanning confocal microscope from Carl Zeiss (Jena, Germany) according to the previous publication .

Techniques: Translocation Assay, Plasmid Preparation, Control, Immunofluorescence, Imaging, Western Blot, Isolation, TUNEL Assay, Staining, Software, Immunostaining

Journal: International Journal of Biological Sciences

Article Title: SUMOylation of Nuclear γ-Actin by SUMO2 supports DNA Damage Repair against Myocardial Ischemia-Reperfusion Injury

doi: 10.7150/ijbs.74407

Figure Lengend Snippet: Site-directed mutagenesis of SUMOylation sites (i.e., K 68 , K 284 ) and subsequent impact on the nuclear translocation of γ-actin and DNA damage in H9c2 cell model. (A) Prediction and site-directed mutagenesis of SUMOylation sites in γ-actin. The lysine residues K 68 and K 284 in γ-actin were predicted as SUMOylation sites by SUMOplot Analysis program ( https://www.abcepta.com/sumoplot ). Lentiral plasmid pCDH-CMV-ACTG-wt for wild type γ-actin was converted to pCDH-CMV-ACTG-mu for mutated γ-actin (K 68 R/K 284 R) by site-directed mutagenesis. (B) Immunofluorescence imaging of intracellular γ-actin and SUMO2. H9c2 cells were transiently tranfected with pdsRED-SUMO2 with pEGFP-ACTG-wt or pEGFP-ACTG-mu. After H/R treatment, the transfected H9c2 cells were analyzed by immunofluorescence imaging for EGFP and dsRED. Scale bar: 50 µm. (C) TUNEL staining of the transfected H9c2 cells. pCDH-CMV-ACTG-wt and pCDH-CMV-ACTG-mu were incorporated into the corresponding lentirus particles for infecting H9c2 cells. and After H/R challenge, stable H9c2 cells (i.e, ACTG1-wt, ACTG1-mu) were stained with TUNEL staining. The cell nuclei were stained with DAPI. The images were quantified by ImageJ software. Scale bar: 100 µm. (D) Immunostaining of 8-OHdG. After H/R challenge, stable H9c2 cells (i.e, ACTG1-wt, ACTG1-mu) were probed with anti-8-OHdG antibody and visualised with Alexa Fluor 647-conjugated secondary antibody. The cell nuclei were stained with DAPI. The images were quantified by ImageJ software. Scale bar: 100 µm. (E) Western blot analysis of p-γ-H2ax. After H/R challenge, stable H9c2 cells (i.e, ACTG1-wt, ACTG1-mu) were analyzed by Western blotting with anti-p-γ-H2ax antibody. The blots were quantified by a densitometric method. The results were shown as mean ± SD (n = 3), * p < 0.05.

Article Snippet: The bound antibodies were detected with Alexa Fluor 488/647 anti-mouse IgG secondary antibody at room temperature for 2 h. After three washes with PBS, the cell nuclei were stained with DAPI for 5 min. Immunofluorescence co-localization analysis was performed on LSM 980 laser scanning confocal microscope from Carl Zeiss (Jena, Germany) according to the previous publication .

Techniques: Mutagenesis, Translocation Assay, Plasmid Preparation, Immunofluorescence, Imaging, Transfection, TUNEL Assay, Staining, Software, Immunostaining, Western Blot