Journal: Aging Cell

Article Title: Serpine 1 induces alveolar type II cell senescence through activating p53‐p21‐Rb pathway in fibrotic lung disease

doi: 10.1111/acel.12643

Figure Lengend Snippet: Knockdown of PAI ‐1 with PAI ‐1 si RNA /sh RNA reduces p53 and p21 protein levels, increases Rb phosphorylation, and attenuates bleomycin‐induced L2 cell senescence. Rat ATII (L2) cells were treated with 50 mU /mL bleomycin for 24 h (A & B) and then cultured in bleomycin‐free medium for additional 72 h (B). (C) L2 cell was transfected with PAI ‐1 si RNA or nontarget si RNA ( NT si RNA ). D‐N) PAI ‐1 sh RNA or NT sh RNA stably transfected L2 cells were treated with bleomycin for 24 h and then cultured in bleomycin‐free medium for additional 24 (D–L) or 72 (M and N) hours. PAI ‐1, serine‐18 phosphorylated p53 (p53 S−18P ), p53, p21, and phosphorylated Rb (ppRb) proteins were determined by Westerns. β‐Actin is used as loading control. D, representative Western blotting pictures; E‐J, semi‐quantified band intensities normalized by β‐actin. (K and L) Immunostaining and quantification of proliferating cell nuclear antigen ( PCNA ). (M and N) SA ‐β‐gal activity revealed by X‐gal staining. α, Significantly different from corresponding saline‐treated cells; β, significantly different from bleomycin‐treated NT sh RNA ‐transfected cells; ζ, significantly different from corresponding NT sh RNA ‐transfected cells ( P < 0.05, n = 3–5).

Article Snippet: To reveal PAI‐1, p53, and p21 proteins in mouse lung tissue, double immunostaining was conducted using formalin‐fixed, paraffin‐embedded tissue slides with antibodies to mouse PAI‐1 (Molecular Innovations, Cat No MA‐33H1F7), p53 (Ancell, Cat No 227‐020), or p21 (Santa Cruz, Cat No SC‐6246) and rabbit polyclonal anti‐mouse proSP‐C antibody (Millipore, Cat No AB3786), following the protocol as we have described previously (El‐Deiry et al ., ).

Techniques: Cell Culture, Transfection, Stable Transfection, Western Blot, Immunostaining, Activity Assay, Staining

Journal: Aging Cell

Article Title: Serpine 1 induces alveolar type II cell senescence through activating p53‐p21‐Rb pathway in fibrotic lung disease

doi: 10.1111/acel.12643

Figure Lengend Snippet: Inhibition of PAI ‐1 activity with a small molecule PAI ‐1 inhibitor TM 5275 attenuates bleomycin‐induced L2 cell senescence. L2 cells were treated with 50 mU /mL bleomycin in the presence or absence of 25 μ m of TM 5275 for 24 hours and then cultured in bleomycin‐free medium for additional 72 (A and B) or 24 (C–G) hours. (A and B) SA ‐β‐gal activity was revealed by X‐gal staining; (C–G) Western analyses of the proteins of interested in cell lysates, the band intensities semi‐quantified by ImageJ software, and normalized by β‐actin. α, Significantly different from corresponding saline‐treated cells; β, significantly different from bleomycin‐treated vehicle controls ( P < 0.05, n = 3).

Article Snippet: To reveal PAI‐1, p53, and p21 proteins in mouse lung tissue, double immunostaining was conducted using formalin‐fixed, paraffin‐embedded tissue slides with antibodies to mouse PAI‐1 (Molecular Innovations, Cat No MA‐33H1F7), p53 (Ancell, Cat No 227‐020), or p21 (Santa Cruz, Cat No SC‐6246) and rabbit polyclonal anti‐mouse proSP‐C antibody (Millipore, Cat No AB3786), following the protocol as we have described previously (El‐Deiry et al ., ).

Techniques: Inhibition, Activity Assay, Cell Culture, Staining, Western Blot, Software

Journal: Aging Cell

Article Title: Serpine 1 induces alveolar type II cell senescence through activating p53‐p21‐Rb pathway in fibrotic lung disease

doi: 10.1111/acel.12643

Figure Lengend Snippet: Knockdown of PAI ‐1 protein with PAI ‐1 sh RNA attenuates doxorubicin‐induced L2 cell senescence. PAI ‐1 sh RNA or NT sh RNA stably transfected L2 cells were treated with 50 n m of doxorubicin (Dox)/saline for 24 h and cultured in doxorubicin‐free medium for additional 24 h (A–D) or 72 h (E and F). PAI ‐1, p53, p21, and β‐actin in cell lysates were determined by Westerns. A, representative Western blotting pictures; B–D, semi‐quantified band intensities by ImageJ program and normalized by β‐actin. E and F, SA ‐β‐gal activity was revealed by X‐gal staining. α, Significantly different from the corresponding saline‐treated cells; β, significantly different from doxorubicin‐treated NT sh RNA ‐transfected cells; ζ, significantly different from saline‐treated NT sh RNA ‐transfected cells ( P < 0.05, n = 3–5).

Article Snippet: To reveal PAI‐1, p53, and p21 proteins in mouse lung tissue, double immunostaining was conducted using formalin‐fixed, paraffin‐embedded tissue slides with antibodies to mouse PAI‐1 (Molecular Innovations, Cat No MA‐33H1F7), p53 (Ancell, Cat No 227‐020), or p21 (Santa Cruz, Cat No SC‐6246) and rabbit polyclonal anti‐mouse proSP‐C antibody (Millipore, Cat No AB3786), following the protocol as we have described previously (El‐Deiry et al ., ).

Techniques: Stable Transfection, Transfection, Cell Culture, Western Blot, Activity Assay, Staining

Journal: Aging Cell

Article Title: Serpine 1 induces alveolar type II cell senescence through activating p53‐p21‐Rb pathway in fibrotic lung disease

doi: 10.1111/acel.12643

Figure Lengend Snippet: Knockdown of p53 protein with p53 si RNA abrogates PAI ‐1 protein‐mediated L2 cell senescence. (A and B) L2 cells were treated with 1 μg/mL of hPAI ‐1, dissolved in 0.1% BAS , or 0.1% bovine serum albumin ( BSA ) for 72 h. (A) PAI ‐1 mRNA was determined by real‐time PCR ; (B) Proteins of interest were determined by Westerns. (C–I) L2 cells were transfected with p53 si RNA or nontarget si RNA ( NT si RNA ) and then treated with hPAI ‐1 or BSA for 72 h. (C and D) SA ‐β‐gal activity was measured by X‐gal staining. (E–I) Western analyses of proteins of interest; E, representative Western blotting pictures; F–I, semi‐quantified band intensities normalized by β‐actin. α, Significantly different from corresponding 0.1% BSA (solvent) controls; β, significantly different from hPAI ‐1‐treated NT si RNA ‐transfected cells; ζ, significantly different from BSA ‐treated NT si RNA ‐transfected cells ( P < 0.05, n = 3–5).

Article Snippet: To reveal PAI‐1, p53, and p21 proteins in mouse lung tissue, double immunostaining was conducted using formalin‐fixed, paraffin‐embedded tissue slides with antibodies to mouse PAI‐1 (Molecular Innovations, Cat No MA‐33H1F7), p53 (Ancell, Cat No 227‐020), or p21 (Santa Cruz, Cat No SC‐6246) and rabbit polyclonal anti‐mouse proSP‐C antibody (Millipore, Cat No AB3786), following the protocol as we have described previously (El‐Deiry et al ., ).

Techniques: Real-time Polymerase Chain Reaction, Transfection, Activity Assay, Staining, Western Blot

Journal: Aging Cell

Article Title: Serpine 1 induces alveolar type II cell senescence through activating p53‐p21‐Rb pathway in fibrotic lung disease

doi: 10.1111/acel.12643

Figure Lengend Snippet: Knockout of the PAI ‐1 gene specifically in ATII cells in mice attenuates bleomycin‐induced ATII cell senescence in vivo . (A and B) Double immunostaining of isolated ATII cells with anti‐ PAI ‐1 and anti‐p53 antibodies. (C and D) Double immunostaining of isolated ATII cells with anti‐p21 and anti‐ SPC antibodies. (E and F) SA ‐β‐gal activity in freshly isolated mouse ATII cells was revealed by X‐gal staining. Left panels are representative SA ‐β‐gal staining pictures; right panel is quantitative data. (G–M) Western analyses of the proteins of interest in isolated ATII cells. (N–S) Double‐immunofluorescence staining of mouse lung tissues with PAI ‐1, p53, or p21 and ATII cell marker SPC . Top panels are representative Western blotting pictures, and bottom panels are quantitative data. α, Significantly different from same genotype, saline‐treated mice; β, significantly different from bleomycin‐treated PAI ‐1 fl/fl mice; ζ, significantly different from saline‐treated PAI ‐1 fl/fl mice ( P < 0.05, n = 3–6).

Article Snippet: To reveal PAI‐1, p53, and p21 proteins in mouse lung tissue, double immunostaining was conducted using formalin‐fixed, paraffin‐embedded tissue slides with antibodies to mouse PAI‐1 (Molecular Innovations, Cat No MA‐33H1F7), p53 (Ancell, Cat No 227‐020), or p21 (Santa Cruz, Cat No SC‐6246) and rabbit polyclonal anti‐mouse proSP‐C antibody (Millipore, Cat No AB3786), following the protocol as we have described previously (El‐Deiry et al ., ).

Techniques: Knock-Out, In Vivo, Double Immunostaining, Isolation, Activity Assay, Staining, Western Blot, Double Immunofluorescence Staining, Marker

Journal: Aging Cell

Article Title: Serpine 1 induces alveolar type II cell senescence through activating p53‐p21‐Rb pathway in fibrotic lung disease

doi: 10.1111/acel.12643

Figure Lengend Snippet: Deletion of the PAI ‐1 gene specifically in ATII cells in mice attenuates bleomycin‐induced lung fibrosis. (A) Body weight changes before and 14 days after bleomycin/saline treatment. (B) The amount of PAI ‐1 protein in BAL fluid measured by ELISA . (C) Trichrome staining of collagen and (D) Sirius red staining of collagen. (E) Hydroxyproline content in mouse lung measured using the Hydroxyproline Assay Kit (Chrondrex, Inc) and expressed as % of hydroxyproline in saline‐treated fl/fl mice. (F–I) Western analyses of procollagen 1α1, procollagen 1α2, and alpha‐smooth muscle actin (α‐ SMA ) in mouse lung tissue. α, Significantly different from same genotype, saline‐treated mice; β, significantly different from bleomycin‐treated PAI ‐1 fl/fl mice ( P < 0.05, n = 3–8).

Article Snippet: To reveal PAI‐1, p53, and p21 proteins in mouse lung tissue, double immunostaining was conducted using formalin‐fixed, paraffin‐embedded tissue slides with antibodies to mouse PAI‐1 (Molecular Innovations, Cat No MA‐33H1F7), p53 (Ancell, Cat No 227‐020), or p21 (Santa Cruz, Cat No SC‐6246) and rabbit polyclonal anti‐mouse proSP‐C antibody (Millipore, Cat No AB3786), following the protocol as we have described previously (El‐Deiry et al ., ).

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

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Drug Targeting of Plasminogen Activator Inhibitor-1 Inhibits Metabolic Dysfunction and Atherosclerosis in a Murine Model of Metabolic Syndrome

doi: 10.1161/ATVBAHA.119.313775

Figure Lengend Snippet: Ldlr−/− mice were fed WD containing or lacking PAI-1 inhibitor for 12 weeks, after which aortic root atherosclerotic plaque composition was assessed. (A) PAI-039 decreases fibrous cap macrophage content. Quantified data (n=5/group; *P<0.05) and representative images demonstrating macrophage invasion (brown color) are shown. (B) MDI-2268 decreases fibrous cap macrophage content. Quantified data (n=6–7/group; *P<0.05) and representative images are shown. (C) MDI-2268 does not significantly affect intimal SMC content, assessed by SMC-α actin immunostaining (brown color). Quantified data (n=4–6/group, difference between groups did not achieve statistical significance; P>0.6) and representative images are shown. (D) MDI-2268 does not significantly affect plaque collagen content, assessed by picrosirius red (PSR) staining. Quantified data (n=6–7/group, difference between groups did not achieve statistical significance; P>0.08) and representative images are shown. L, lumen.

Article Snippet: Plasma PAI-1 antigen was measured either by a Luminex multiplex assay 39 or a standard ELISA utilizing anti-murine PAI-1 capture antibody (Molecular Innovations clone H34G6; coating concentration 1 μg/mL) and biotinylated anti-murine PAI-1 detection antibody (Molecular Innovations, ASMPAI-GF-BIO, 1 μg/mL).

Techniques: Immunostaining, Staining

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Drug Targeting of Plasminogen Activator Inhibitor-1 Inhibits Metabolic Dysfunction and Atherosclerosis in a Murine Model of Metabolic Syndrome

doi: 10.1161/ATVBAHA.119.313775

Figure Lengend Snippet: (A) SMCs were incubated for 24 hours with recombinant PAI-1 (10 μg/mL), recombinant PAI-1 and PAI-039 (25 μM), or vehicle control, after which SA-βGal expression (% positive cells) was measured; n= 5–7/group; *P<0.05 vs. other groups. (B) SMCs (passage number 6–8) were incubated 12 hours with or without anti-LRP1 antibody (at indicated concentrations [μg/mL]), after which PAI-1 (1 μg/mL, “+”) or vehicle control (“-”) was added. Cells were incubated an additional 24 hours, after which SA-βGal expression was measured; n=5/group, *P<0.05 vs. control (untreated SMCs). (C) SMCs (passage number 7) were incubated 24 hours with PAI-1-I91L (recombinant PAI-1 mutant with increased stability, normal anti-protease activity, and normal LRP1-binding affinity, concentration 1 μg/mL), PAI-1-I91L,K80/207A (recombinant PAI-1 mutant with increased stability, normal anti-protease activity, and a greater than 20-fold reduction in LRP1-binding affinity; concentration 1 μg/mL), or vehicle control, after which SA-βGal expression was measured; n=4/group; *P<0.001 vs. other groups.

Article Snippet: Plasma PAI-1 antigen was measured either by a Luminex multiplex assay 39 or a standard ELISA utilizing anti-murine PAI-1 capture antibody (Molecular Innovations clone H34G6; coating concentration 1 μg/mL) and biotinylated anti-murine PAI-1 detection antibody (Molecular Innovations, ASMPAI-GF-BIO, 1 μg/mL).

Techniques: Incubation, Recombinant, Expressing, Mutagenesis, Activity Assay, Binding Assay, Concentration 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: 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: Nucleic Acids Research

Article Title: Characterisation of aptamer–target interactions by branched selection and high-throughput sequencing of SELEX pools

doi: 10.1093/nar/gkv700

Figure Lengend Snippet: An overview of the method for parallelised aptamer characterisation. After five rounds of selection for binding to the serpin PAI-1, the enriched RNA pool was subjected to one round of parallel selections for binding to different PAI-1 variants (wild type and single-residue alanine mutants) and conformers (active and latent form) as well as PAI-1 presented by different antibodies or the natural extracellular matrix protein, vitronectin (VN). After a washing step, the retained RNA was reverse transcribed, PCR amplified with barcoded primers, and sequenced in one reaction by Illumina sequencing.

Article Snippet: Wells of 96-well microtiter Maxisorp plates (NUNC) were coated overnight with a rabbit polyclonal anti-PAI-1 antibody (5 μg/ml), the monoclonal anti-PAI-1 antibody mAb-1 (5 μg/ml) binding in α-helix C , or monomeric vitronectin (1 μg/ml; Molecular Innovations) using 0.1 M Na 2 CO 3 /NaHCO 3 pH 9.6.

Techniques: Selection, Binding Assay, Amplification, Sequencing

Journal: Nucleic Acids Research

Article Title: Characterisation of aptamer–target interactions by branched selection and high-throughput sequencing of SELEX pools

doi: 10.1093/nar/gkv700

Figure Lengend Snippet: Binding sites of aptamers paionap-5 and -40 on PAI-1. Left panel: PAI-1 in a cartoon representation with residues important for binding of the aptamers shown as red (Arg78, Lys82, Phe116 and Arg120 recognised by both paionap-5 and -40) and yellow spheres (Lys124 recognised only by paionap-40) . Middle panel: Surface representation model of PAI-1 using same colour codes. Residues in blue (Lys71 and Tyr81) are unimportant for aptamer binding and were included in the branched selection-HTS analysis. Right panel: The position of the somatomedin B domain of vitronectin (green) when bound to PAI-1. The figure was prepared using PyMOL (the PyMOL Molecular Graphics System, Version 1.5.0.4 Schrödinger, LLC) and PDB ID: 1OC0.

Article Snippet: Wells of 96-well microtiter Maxisorp plates (NUNC) were coated overnight with a rabbit polyclonal anti-PAI-1 antibody (5 μg/ml), the monoclonal anti-PAI-1 antibody mAb-1 (5 μg/ml) binding in α-helix C , or monomeric vitronectin (1 μg/ml; Molecular Innovations) using 0.1 M Na 2 CO 3 /NaHCO 3 pH 9.6.

Techniques: Binding Assay, Selection

Journal: Nucleic Acids Research

Article Title: Characterisation of aptamer–target interactions by branched selection and high-throughput sequencing of SELEX pools

doi: 10.1093/nar/gkv700

Figure Lengend Snippet: Evaluation of the PAI-1 mutant specificity of the thousand most prevalent sequences of the input RNA pool. For each single-residue alanine mutant used in the HTS-microplate assay a plot of enrichment factors for wild type (EF wt ; x-axis) versus mutant (EF variant ; y-axis) is shown. The x-axis crosses the y-axis at EF variant = 1 and the y-axis crosses the x-axis at EF wt = 1.

Article Snippet: Wells of 96-well microtiter Maxisorp plates (NUNC) were coated overnight with a rabbit polyclonal anti-PAI-1 antibody (5 μg/ml), the monoclonal anti-PAI-1 antibody mAb-1 (5 μg/ml) binding in α-helix C , or monomeric vitronectin (1 μg/ml; Molecular Innovations) using 0.1 M Na 2 CO 3 /NaHCO 3 pH 9.6.

Techniques: Mutagenesis, Variant Assay

Journal: Nucleic Acids Research

Article Title: Characterisation of aptamer–target interactions by branched selection and high-throughput sequencing of SELEX pools

doi: 10.1093/nar/gkv700

Figure Lengend Snippet: Secondary structure prediction for RNA variants binding to the PAI-1:vitronectin complex. LocARNA alignment of the ten variants of SEQ ID 58088 ( A ) and SEQ ID 415 ( B ) after one round of selection for binding to the PAI-1:vitronectin complex. ( C and D ) The predicted secondary structures for SEQ ID 58088 and SEQ ID 415, respectively, based on the LocARNA alignment. See the legend to Figure for details.

Article Snippet: Wells of 96-well microtiter Maxisorp plates (NUNC) were coated overnight with a rabbit polyclonal anti-PAI-1 antibody (5 μg/ml), the monoclonal anti-PAI-1 antibody mAb-1 (5 μg/ml) binding in α-helix C , or monomeric vitronectin (1 μg/ml; Molecular Innovations) using 0.1 M Na 2 CO 3 /NaHCO 3 pH 9.6.

Techniques: Binding Assay, Selection