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: 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