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: The American Journal of Pathology

Article Title: A Protein C Deficiency Exacerbates Inflammatory and Hypotensive Responses in Mice During Polymicrobial Sepsis in a Cecal Ligation and Puncture Model

doi:

Figure Lengend Snippet: Plasma coagulation factor assays of WT and PC+/− mice after CLP surgery. a: Plasma PC levels. The plasma PC levels were determined by ELISA before (WT, n = 4; PC+/−, n = 5), and 6 hours (WT, n = 5; PC+/−, n = 7) and 24 hours (WT, n = 5; PC+/−, n = 5) after CLP surgery in WT and PC+/− male mice. *, P < 0.001; #, P < 0.01. b: Plasma fibrinogen levels were determined by a coagulometric assay at 6 hours (WT, n = 4; PC+/−, n = 5) and 24 hours (WT, n = 15; PC+/−, n = 14) after, CLP surgery and in resting (WT, n = 11; PC+/−, n = 9) WT and PC+/− male mice. *, P = 0.016. c: Plasma PAI-1 levels were determined by ELISA before (WT, n = 3; PC+/−, n = 4), and 6 hours (WT, n = 3; PC+/−, n = 7) and 24 hours (WT, n = 5; PC+/−, n = 4) after, CLP surgery in WT and PC+/− male mice. d: Plasma FXII levels relative to the resting WT group, as measured by a coagulometric assay, before (WT, n = 4; PC+/−, n = 3) and 24 hours after (WT, n = 5; PC+/−, n = 3), CLP surgery. *, P = 0.05 (Kruskal-Wallis). e: Plasma FXI levels relative to the resting WT group as measured by a coagulometric assay, before (WT, n = 7; PC+/−, n = 5) and 24 hours after (WT, n = 5; PC+/−, n = 3), CLP surgery. No significant differences were observed.

Article Snippet: After four washes with PBS/Tween-20, 100 μl of 4 μg/ml rabbit anti-mouse PAI-1 polyclonal antibody (Molecular Innovations) in PBS was added to the wells and incubated for 2 hours.

Techniques: Coagulation, Enzyme-linked Immunosorbent Assay

Journal: The American Journal of Pathology

Article Title: A Protein C Deficiency Exacerbates Inflammatory and Hypotensive Responses in Mice During Polymicrobial Sepsis in a Cecal Ligation and Puncture Model

doi:

Figure Lengend Snippet: Gene expression ratio to RPL-19 of PAI-1 (a), MPO (b), IL-1β (c), and IL-6 (d) relative to the WT resting group (n = 3) in liver, kidney, and lung, as determined by RT-PCR in WT mice 24 hours after CLP (n = 3), PC+/− resting mice (n = 3), and PC+/− mice 24 hours after CLP (n = 4). a: In liver, PC+/− mice 24 hours after CLP were different from PC+/− resting and WT resting groups (P = 0.025, K-W), in kidney, differences between groups were not significant (K-W, multicomparison test). b: In liver, PC+/− resting mice were different from PC+/− mice 24 hours after CLP and WT resting groups (P = 0.055); in kidney, PC+/− mice 24 hours after CLP were different from all of the other groups (P = 0.02, K-W); in lung, PC+/− resting mice were different from PC+/− and WT mice 24 hours after CLP (P = 0.08, K-W). c: In liver, no significant differences between groups were found; in kidney: PC+/− group 24 hours after CLP was different from WT and PC+/− resting groups (P = 0.04, K-W); in lung: the PC+/− group after CLP was not different form the PC+/− resting group (T-K); P = not significant (K-W). d: In liver, PC+/− mice 24 hours after CLP were different from the WT resting group (P = 0.11, K-W); in kidney, PC+/− mice 24 hours after CLP were different from the WT and PC+/− resting groups (P = 0.032, K-W); in lung: no significant differences between the groups were found (K-W).

Article Snippet: After four washes with PBS/Tween-20, 100 μl of 4 μg/ml rabbit anti-mouse PAI-1 polyclonal antibody (Molecular Innovations) in PBS was added to the wells and incubated for 2 hours.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Cells

Article Title: Annexin A2 Egress during Calcium-Regulated Exocytosis in Neuroendocrine Cells

doi: 10.3390/cells9092059

Figure Lengend Snippet: Tissue plasminogen activator (t-PA) is present on the outer leaflet of the plasma membrane of stimulated chromaffin cells. ( a ) Dual labeling of cell surface t-PA and exocytotic sites. Cells were stimulated with nicotine 20 µM in the presence of anti-t-PA and anti-DBH antibodies. Cells were then fixed and incubated with secondary antibodies coupled to Alexa Fluor ® 488 and Alexa Fluor ® 551, respectively. Confocal images were recorded in the same optical section and with the same parameters of lasers and photomultipliers. Scale bar: 10 µM. ( b ) The t-PA labeling on the surface of chromaffin cells in the resting condition (R), stimulated with 20 µM nicotine without (S) or after EGTA wash (S + EGTA). Anti-t-PA antibodies were revealed with Alexa Fluor ® 488-conjugated anti-rabbit antibodies and F-actin with TRITC-phalloidin to visualize the cell shape. Confocal images were recorded in the same optical section by a dual exposure procedure. Scale bar: 10 µM. ( c ) Semi-quantitative analysis of t-PA labeling on the cell surface of chromaffin cells in the resting condition (R), stimulated with 20 µM nicotine (S) without (Control) or after EGTA wash (S + EGTA). The t-PA labeling is expressed in arbitrary units. Statistical significance for medians was determined using a Mann-Whitney test. Dotted lines indicate the mean and asterisks statistical significance (*** = p < 0.001, ** = p < 0.01). Three experiments were done on independent cell cultures and pooled ( n = 36 and 65 control cells, 40 and 75 EGTA-treated cells for resting and stimulated conditions, respectively). ( d ) Lysate, secreted material and EGTA eluate from chromaffin cells in the resting condition (R) or stimulated with nicotine 20 µM (S) were analyzed by western blot and revealed with anti-t-PA and anti-AnxA2 antibodies. Data correspond to a typical experiment representative of three independent experiments.

Article Snippet: Rabbit polyclonal anti-mouse tissue plasminogen activator (t-PA) antibodies were from Molecular Innovations (Novi, MI, USA).

Techniques: Labeling, Incubation, MANN-WHITNEY, Western Blot

Journal: Cells

Article Title: Annexin A2 Egress during Calcium-Regulated Exocytosis in Neuroendocrine Cells

doi: 10.3390/cells9092059

Figure Lengend Snippet: Membrane topography of AnxA2, t-PA and exocytotic sites after immunogold labeling of the outer face of the plasma membrane sheets prepared from stimulated chromaffin cells. ( a ) Plasma membrane sheets were prepared from bovine chromaffin cells stimulated by nicotine for 5 min. To label DBH, AnxA2 and t-PA exposed at the surface of cells undergoing exocytosis, anti-DBH, anti-AnxA2 and anti-t-PA antibodies were added during stimulation. Membrane sheets were labeled with anti-mouse antibodies coupled to 10 nm gold particles to detect DBH antibodies revealing exocytotic sites (red circle) and rabbit antibodies coupled to 15 nm gold particles to label AnxA2 or t-PA (green circle). ( b ) The histogram represents the relative distribution of 15 nm gold particles as a function of their distance from the granule membrane once inserted in the plasma membrane (blue line). The distance was measured and the number of particles was counted manually with Photoshop. Three experiments were done on independent cell cultures ( c ). Double staining experiment for t-PA (10 nm gold particles) and AnxA2 (15 nm gold particles) were performed with the same protocol. Scale bar: 100 nm. ( d ) The histogram represents the relative distribution of 10 nm gold particles (t-PA) as a function of their distance from 15 nm gold particles (AnxA2). The distance was measured and the number of particles was counted manually with Photoshop. Two experiments were done on two independent cell cultures.

Article Snippet: Rabbit polyclonal anti-mouse tissue plasminogen activator (t-PA) antibodies were from Molecular Innovations (Novi, MI, USA).

Techniques: Labeling, Double Staining