Journal: Journal of xenobiotics

Article Title: Assessment of the In Vitro Phosphatidylinositol Glycan Class A (PIG-A) Gene Mutation Assay Using Human TK6 and Mouse Hepa1c1c7 Cell Lines.

doi: 10.3390/jox14030073

Figure Lengend Snippet: Figure 1. Mutant cell enrichment by separation with anti-CD59 microbeads and paramagnetic separator.

Article Snippet: Anti-mouse PE-conjugated CD59 antibody (Clone REA287, Lot no. 5230105732), anti-human PE-conjugated CD59 antibody (Clone REA496, Lot no. 1322120604), lyophilized anti-PE MicroBeads (Lot no. 5181221156), LS columns (Lot no. 5180927085), and a QuadroMACS separator were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany).

Techniques: Mutagenesis

Journal: Journal of xenobiotics

Article Title: Assessment of the In Vitro Phosphatidylinositol Glycan Class A (PIG-A) Gene Mutation Assay Using Human TK6 and Mouse Hepa1c1c7 Cell Lines.

doi: 10.3390/jox14030073

Figure Lengend Snippet: Figure 8. PIG-A gating strategy: (a) gating strategies for separating GPI(−) (lower left region) and GPI(+) (lower right region) of TK6 cell line; (b) gating strategies for separating GPI(−) (lower left region) and GPI(+) (lower right region) of Hepa1c1c7 cell line. PIG-A gene mutated cells were not stained with anti-CD59 antibodies and had no PE signal that was conjugated with antibodies.

Article Snippet: Anti-mouse PE-conjugated CD59 antibody (Clone REA287, Lot no. 5230105732), anti-human PE-conjugated CD59 antibody (Clone REA496, Lot no. 1322120604), lyophilized anti-PE MicroBeads (Lot no. 5181221156), LS columns (Lot no. 5180927085), and a QuadroMACS separator were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany).

Techniques: Staining

Journal: International Journal of Molecular Sciences

Article Title: Attenuation of Ventilation-Enhanced Epithelial–Mesenchymal Transition through the Phosphoinositide 3-Kinase-γ in a Murine Bleomycin-Induced Acute Lung Injury Model

doi: 10.3390/ijms24065538

Figure Lengend Snippet: Inhibition of lung stretch-mediated oxidative stress, PAI-1, and VEGF production by AS605240. Five days after administering bleomycin, ( A ) MDA, ( B ) TAC, ( C ) PAI-1, and ( D ) VEGF secretion in BAL fluid from the lungs of nonventilated control mice and those subjected to a tidal volume at 6 or 30 mL/kg for 5 h with room air ( n = 5 per group). AS605240 5 mg/kg was given intraperitoneally 1 h before ventilation. * p < 0.05 versus the nonventilated control mice with bleomycin pretreatment; † p < 0.05 versus all other groups. BAL = bronchoalveolar lavage; MDA = malondialdehyde; PAI-1 = plasminogen activator inhibitor-1; TAC = total antioxidant capacity; and VEGF = vascular endothelial growth factor.

Article Snippet: PAI-1 (0.02 ng/mL) and VEGF (1.8 pg/mL) were detected in serum and bronchoalveolar lavage fluid using immunoassay kits containing primary polyclonal anti-mouse antibodies that were cross-reactive with rat and mouse PAI-1 and VEGF (VEGF: Biosource International, Camarillo, CA, USA; PAI-1: Molecular Innovations Inc., Plymouth Meeting, PA, USA).

Techniques: Inhibition

Journal: International Journal of Molecular Sciences

Article Title: Attenuation of Ventilation-Enhanced Epithelial–Mesenchymal Transition through the Phosphoinositide 3-Kinase-γ in a Murine Bleomycin-Induced Acute Lung Injury Model

doi: 10.3390/ijms24065538

Figure Lengend Snippet: Reduction of lung stretch-induced lung inflammation in PI3K-γ-deficient mice. ( A ) Evans blue dye analysis, ( B ) lung wet-to-dry-weight ratio), ( C ) PaO 2 /FiO 2 , ( D ) enhanced pause, ( E ) MDA, ( F ) TAC, ( G ) PAI-1, and ( H ) VEGF from the lungs of nonventilated control mice and those subjected to a tidal volume of 30 mL/kg for 5 h with bleomycin administration ( n = 5 per group). * p < 0.05 versus the nonventilated control mice with bleomycin; and † p < 0.05 versus PI3K-γ-deficient mice.

Article Snippet: PAI-1 (0.02 ng/mL) and VEGF (1.8 pg/mL) were detected in serum and bronchoalveolar lavage fluid using immunoassay kits containing primary polyclonal anti-mouse antibodies that were cross-reactive with rat and mouse PAI-1 and VEGF (VEGF: Biosource International, Camarillo, CA, USA; PAI-1: Molecular Innovations Inc., Plymouth Meeting, PA, USA).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Attenuation of Ventilation-Enhanced Epithelial–Mesenchymal Transition through the Phosphoinositide 3-Kinase-γ in a Murine Bleomycin-Induced Acute Lung Injury Model

doi: 10.3390/ijms24065538

Figure Lengend Snippet: Schematic figure illustrating the signaling pathway activation with mechanical ventilation and bleomycin. Bleomycin-induced augmentation of mechanical stretch-mediated cytokine production and lung damage were attenuated in PI3K-γ-deficient mice and pharmacological inhibition with AS605240. EMT = epithelial–mesenchymal transition; ROS = reactive oxygen species; PAI-1 = plasminogen activator inhibitor-1; PI3K-γ -/- = PI3K-γ-deficient mice; VEGF = vascular endothelial growth factor; and VILI = ventilator-induced lung injury.

Article Snippet: PAI-1 (0.02 ng/mL) and VEGF (1.8 pg/mL) were detected in serum and bronchoalveolar lavage fluid using immunoassay kits containing primary polyclonal anti-mouse antibodies that were cross-reactive with rat and mouse PAI-1 and VEGF (VEGF: Biosource International, Camarillo, CA, USA; PAI-1: Molecular Innovations Inc., Plymouth Meeting, PA, USA).

Techniques: Activation Assay, Inhibition

Journal: Aging and Disease

Article Title: Aging, Plasminogen Activator Inhibitor 1, Brain Cell Senescence, and Alzheimer’s Disease

doi: 10.14336/AD.2022.1220

Figure Lengend Snippet: PAI-1 mediates H 2 O 2 -induced senescence in primary human and mouse astrocytes. (A-D) Western analyses and quantification of PAI-1, p53, and p21 in primary human astrocytes transfected with PAI-1siRNA/NTsiRNA and then treated with 200 μM H 2 O 2 /saline. The results were normalized by GAPDH (n=3). (E, F) X-gal staining and quantification of SA-β-gal positive astrocytes (n=5). The results were expressed as percentages of total cells. (G, H) X-gal staining of SA-β-gal positive astrocytes (n=3). The results were expressed as percentage of total cells. (I-L) Western analysis and quantifications of PAI-1 and p21 proteins. The band intensities presented were normalized by GAPDH. (M-P) Western analyses and quantifications of PAI-1, p16, and pRb proteins in primary astrocytes isolated from wild type or PAI-1 -/- mice treated with H 2 O 2 or saline. The band intensities presented were normalized by GAPDH (n=4-5). (Q, R) ELISAs of IL-6 and IGFBP3 proteins in the culture medium (n=4-6).

Article Snippet: To reveal PAI-1 and p16 (a senescence marker) positive astrocytes in brain tissues of LOAD patients and healthy controls, mouse monoclonal anti-PAI-1 (Molecular Innovations, Cat No MA-33H1F7) or mouse monoclonal anti-p16 (Santa Cruz Biotech, Cat No SC-1661) and rabbit polyclonal anti-GFAP antibody (Sigma-Aldrich, Inc., Cat No G9269, St. Louis, MO USA) were used.

Techniques: Western Blot, Transfection, Staining, Isolation

Journal: Aging and Disease

Article Title: Aging, Plasminogen Activator Inhibitor 1, Brain Cell Senescence, and Alzheimer’s Disease

doi: 10.14336/AD.2022.1220

Figure Lengend Snippet: Overexpression of wild type or secretion deficient PAI-1 induces senescence in primary mouse astrocytes. (A) ELISA of PAI-1 protein in culture medium (CM). (B-E) Western analyses and quantification of PAI-1, p21, and p16 proteins in wtPAI-1, sdPAI-1, and control lentivirus-transduced primary PAI-1 -/- mouse astrocytes. The band intensities presented were normalized by GAPDH (n=3-6). (F, G) X-gal staining and quantification of SA-β-gal positive cells, the results expressed as percentages of total cells. (H) ELISA of IL-6 in the CM (n=6).

Article Snippet: To reveal PAI-1 and p16 (a senescence marker) positive astrocytes in brain tissues of LOAD patients and healthy controls, mouse monoclonal anti-PAI-1 (Molecular Innovations, Cat No MA-33H1F7) or mouse monoclonal anti-p16 (Santa Cruz Biotech, Cat No SC-1661) and rabbit polyclonal anti-GFAP antibody (Sigma-Aldrich, Inc., Cat No G9269, St. Louis, MO USA) were used.

Techniques: Over Expression, Enzyme-linked Immunosorbent Assay, Western Blot, Staining

Journal: Aging and Disease

Article Title: Aging, Plasminogen Activator Inhibitor 1, Brain Cell Senescence, and Alzheimer’s Disease

doi: 10.14336/AD.2022.1220

Figure Lengend Snippet: PAI-1 protein level is increased, in correlation with increases of cell cycle repressors, in the hippocampus/cortex of old SAMP8 mice. (A, B) Western analyses and quantification of PAI-1, p53 and p21 proteins in hippocampus/cortex of 12-month-old wild type and SAMP8 mice. The band intensities were normalized by GAPDH bands (n=4-6). (C) Pearson correlation analysis of PAI-1 protein level and p53/p21 protein level (n=10). (D) ELISA of PAI-1 protein in the hippocampus/cortex of young and old SAMP8 mice (n=4-6). (E, F) Western analyses and quantification of proteins of interest in the hippocampus/cortex of 4-month and 12-month-old SAMP8 mice. The results were expressed as percentage of 4M-old mice. (G-I) Immunostaining and quantifications of senescent astrocytes in hippocampus and cortex of 4-month and 12-month-old SAMP8 mice by double immunostaining of mH2A and GFAP (n=4-6). The results were expressed as percentage of total cells.

Article Snippet: To reveal PAI-1 and p16 (a senescence marker) positive astrocytes in brain tissues of LOAD patients and healthy controls, mouse monoclonal anti-PAI-1 (Molecular Innovations, Cat No MA-33H1F7) or mouse monoclonal anti-p16 (Santa Cruz Biotech, Cat No SC-1661) and rabbit polyclonal anti-GFAP antibody (Sigma-Aldrich, Inc., Cat No G9269, St. Louis, MO USA) were used.

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Immunostaining, Double Immunostaining

Journal: Aging and Disease

Article Title: Aging, Plasminogen Activator Inhibitor 1, Brain Cell Senescence, and Alzheimer’s Disease

doi: 10.14336/AD.2022.1220

Figure Lengend Snippet: PAI-1 protein level is increased in the prefrontal cortex of LOAD patients. (A, B) Western analyses and quantifications of PAI-1, p53, and p21 proteins in the prefrontal cortex of LOAD patients and age-matched healthy controls. The band intensities presented were normalized by GAPDH (n=5-10). (C) Pearson correlation analysis of PAI-1 protein level and p53 protein/p21 protein level (n=15). (D, E) Double immunostaining and quantification of PAI-1 positive astrocytes in cortex slides from LOAD patients and control subjects (n=5-6). The results were expressed as percentages of controls. (F, G) Double immunostaining and quantification of p16 positive astrocytes in cortex slides from LOAD patients and control subjects (n=5-6). The results were expressed as percentages of controls.

Article Snippet: To reveal PAI-1 and p16 (a senescence marker) positive astrocytes in brain tissues of LOAD patients and healthy controls, mouse monoclonal anti-PAI-1 (Molecular Innovations, Cat No MA-33H1F7) or mouse monoclonal anti-p16 (Santa Cruz Biotech, Cat No SC-1661) and rabbit polyclonal anti-GFAP antibody (Sigma-Aldrich, Inc., Cat No G9269, St. Louis, MO USA) were used.

Techniques: Western Blot, Double Immunostaining

Journal: Aging and Disease

Article Title: Aging, Plasminogen Activator Inhibitor 1, Brain Cell Senescence, and Alzheimer’s Disease

doi: 10.14336/AD.2022.1220

Figure Lengend Snippet: Silencing or inhibition of PAI-1 attenuates H 2 O 2 -induced U87 cell senescence. (A, B) Western analyses and quantification of PAI-1, p21, and phosphorylated Rb (pRb) proteins in U87 cells. The band intensities presented were normalized by GAPDH. C) ELISA of PAI-1 protein in the culture medium. (D, E) X-gal staining and quantification of SA-β-gal positive U87 cells. The results were expressed as percentages of total cells. (F, G) Western analyses and quantifications of p53 and p21 proteins in U87 cells. The band intensities presented were normalized by GAPDH. (H, I) X-gal staining of SA-β-gal positive U87 cells (n=3). The results were expressed as percentages of total cells.

Article Snippet: To reveal PAI-1 and p16 (a senescence marker) positive astrocytes in brain tissues of LOAD patients and healthy controls, mouse monoclonal anti-PAI-1 (Molecular Innovations, Cat No MA-33H1F7) or mouse monoclonal anti-p16 (Santa Cruz Biotech, Cat No SC-1661) and rabbit polyclonal anti-GFAP antibody (Sigma-Aldrich, Inc., Cat No G9269, St. Louis, MO USA) were used.

Techniques: Inhibition, Western Blot, Enzyme-linked Immunosorbent Assay, Staining

Journal: Aging and Disease

Article Title: Aging, Plasminogen Activator Inhibitor 1, Brain Cell Senescence, and Alzheimer’s Disease

doi: 10.14336/AD.2022.1220

Figure Lengend Snippet: Astrocyte SASP promotes apoptotic responses in neurons. (A) Flow diagram showing the major steps of the experiments. (B-E) U87 cells were treated with 200 μM H 2 O 2 for 48 hrs. (B) Western analyses of PAI-1, p21, and phosphorylated Rb proteins. (C, D) X-gal staining and quantification of SA-β-gal positive U87 cells (n=3). The results were expressed as percentages of total cells. E) Caspase activities in the conditional medium (CM) of U87 cells (n=3). F-I) SH-SY5Y cells were cultured with the CM from senescent U87 cells for 48 hrs. (F-H) Western analyses and quantifications of Bax and cleaved caspase3 proteins. The band intensities presented were normalized by GAPDH (n=3). (I) Caspase 3/7 activity in the culture medium of SH-SY5Y (n=3). (J-M) Primary mouse neurons were cultured with the CM from primary PAI-1 -/- mouse astrocytes transduced with wtPAI-1, sdPAI-1, or control lentiviruses as described in for 48 hours. (J, K) TUNEL staining of apoptotic neurons (n=6). The results were expressed as percentages of total cell number. (L) ELISA of caspase 3/7 and (M) ELISA of caspase 9 activities in the culture medium of primary neuron (n=4).

Article Snippet: To reveal PAI-1 and p16 (a senescence marker) positive astrocytes in brain tissues of LOAD patients and healthy controls, mouse monoclonal anti-PAI-1 (Molecular Innovations, Cat No MA-33H1F7) or mouse monoclonal anti-p16 (Santa Cruz Biotech, Cat No SC-1661) and rabbit polyclonal anti-GFAP antibody (Sigma-Aldrich, Inc., Cat No G9269, St. Louis, MO USA) were used.

Techniques: Western Blot, Staining, Cell Culture, Activity Assay, Transduction, TUNEL Assay, Enzyme-linked Immunosorbent Assay

Journal: Aging and Disease

Article Title: Aging, Plasminogen Activator Inhibitor 1, Brain Cell Senescence, and Alzheimer’s Disease

doi: 10.14336/AD.2022.1220

Figure Lengend Snippet: Hypothetic mechanism by which PAI-1 promotes brain cell senescence and neuron apoptosis during aging and in LOAD. PAI-1 expression increases with age and in LOAD brain. Increased PAI-1, intracellular or extracellular, leads to increases in the expression of cell cycle repressors p53, p21, and/or p16 as well as senescence in brain cells, including astrocytes. Senescent astrocytes in turn secrete pathologically active molecules, including PAI-1, which induces neuron apoptosis.

Article Snippet: To reveal PAI-1 and p16 (a senescence marker) positive astrocytes in brain tissues of LOAD patients and healthy controls, mouse monoclonal anti-PAI-1 (Molecular Innovations, Cat No MA-33H1F7) or mouse monoclonal anti-p16 (Santa Cruz Biotech, Cat No SC-1661) and rabbit polyclonal anti-GFAP antibody (Sigma-Aldrich, Inc., Cat No G9269, St. Louis, MO USA) were used.

Techniques: Expressing

Journal: International Journal of Molecular Sciences

Article Title: Improvement of Radiotherapy-Induced Lacrimal Gland Injury by Induced Pluripotent Stem Cell-Derived Conditioned Medium via MDK and Inhibition of the p38/JNK Pathway

doi: 10.3390/ijms151018407

Figure Lengend Snippet: iPSC-CM suppressed the RILI-associated inflammatory response. ( A ) Immunohistochemical staining for PAI1 in iPSC-CM treated RILI and normal mouse lacrimal glands. Scale bar = 25 µm; ( B ) Immunohistochemical staining for HMGB1 in iPSC-CM treated RILI and normal mouse lacrimal glands. Scale bar = 25 µm; ( C,D ) Quantification of the mean density of immunohistochemical staining of these sections (CM: iPSC-CM, RT: radiotherapy, MFCM: mouse fibroblasts conditioned medium). N = 5. Values are means ± SEM. * p < 0.01; (E) Neutrophils migrated into the injured gland sites revealed by the neutrophil counts and myeloperoxidase (MPO) assay. * p < 0.01.

Article Snippet: Paraformaldehyde-fixed, paraffin-embedded mouse lacrimal gland sections (4 µm) were first incubated with a primary antibody against mouse PAI1 (Molecular Innovations, Inc., Southfield, MI, USA) and HMGB1 (R&D System Inc., Minneapolis, MN, USA) overnight at 4 °C and then incubated with the goat anti-mouse IgG secondary antibodies (Molecular Innovations, Inc., Southfield, MI, USA).

Techniques: Immunohistochemical staining, Staining, MPO Assay