foxo3a  (Cell Signaling Technology Inc)


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    Structured Review

    Cell Signaling Technology Inc foxo3a
    <t>FoxO3a</t> expression decreases on collagen. A, serum-starved human lung fibroblasts were attached to collagen (100 μg/ml, upper panel)-coated plates as a function of time. Akt and Ser-473-phosphorylated Akt levels were then measured. B, human lung fibroblasts were serum-starved for 2 days followed by attachment to collagen-coated plates as a function of time. FoxO3a, FoxO1, and FoxO4 expression levels were measured (upper and lower panels, respectively). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and actin were used as a loading control. C, serum-starved human lung fibroblasts were attached to fibronectin- (10 μg/ml), vitronectin- (10 μg/ml), and laminin (10 μg/ml)-coated plates as a function of time. FoxO3a expression levels were measured. Actin was used as a loading control.
    Foxo3a, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "?1-Integrin-Collagen Interaction Suppresses FoxO3a by the Coordination of Akt and PP2A"

    Article Title: ?1-Integrin-Collagen Interaction Suppresses FoxO3a by the Coordination of Akt and PP2A

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.052845

    FoxO3a expression decreases on collagen. A, serum-starved human lung fibroblasts were attached to collagen (100 μg/ml, upper panel)-coated plates as a function of time. Akt and Ser-473-phosphorylated Akt levels were then measured. B, human lung fibroblasts were serum-starved for 2 days followed by attachment to collagen-coated plates as a function of time. FoxO3a, FoxO1, and FoxO4 expression levels were measured (upper and lower panels, respectively). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and actin were used as a loading control. C, serum-starved human lung fibroblasts were attached to fibronectin- (10 μg/ml), vitronectin- (10 μg/ml), and laminin (10 μg/ml)-coated plates as a function of time. FoxO3a expression levels were measured. Actin was used as a loading control.
    Figure Legend Snippet: FoxO3a expression decreases on collagen. A, serum-starved human lung fibroblasts were attached to collagen (100 μg/ml, upper panel)-coated plates as a function of time. Akt and Ser-473-phosphorylated Akt levels were then measured. B, human lung fibroblasts were serum-starved for 2 days followed by attachment to collagen-coated plates as a function of time. FoxO3a, FoxO1, and FoxO4 expression levels were measured (upper and lower panels, respectively). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and actin were used as a loading control. C, serum-starved human lung fibroblasts were attached to fibronectin- (10 μg/ml), vitronectin- (10 μg/ml), and laminin (10 μg/ml)-coated plates as a function of time. FoxO3a expression levels were measured. Actin was used as a loading control.

    Techniques Used: Expressing

    FoxO3a activity decreases in response to fibroblast attachment to collagen. A, serum-starved human lung fibroblasts were attached to collagen-coated plates as a function of time and phosphorylated FoxO3a (p-FoxO3a, Ser-253) and actin levels were measured (upper panel). Cells were placed on fibronectin-, vitronectin-, and laminin-coated plates as a function of time and phosphorylated FoxO3a levels were measured (lower panel). Actin was used as a loading control. B, serum-starved human lung fibroblasts were attached at 0 to 100 μg/ml of collagen-coated plates for 30 min. FoxO3a and phosphorylated FoxO3a levels were then measured. Actin was used as a loading control. C, upper panel, human lung fibroblasts were serum-starved followed by preincubation with PI3K or Akt inhibitor as indicated for 45 min. Cells were then allowed to attach to collagen-coated plates for 30 min. FoxO3a expression levels were then measured. Actin was used as a loading control. PI3KI, PI3K inhibitor; AktI, Akt Inhibitor; s/s, serum-starved fibroblasts. DMSO, dimethyl sulfoxide control. Lower panel, fold-changes in FoxO3a expression (FoxO3a/actin) in the presence of PI3K or Akt inhibitor on collagen. The results are representative of three different experiments. D, upper panel, serum-starved lung fibroblasts were preincubated with PI3K (200 nm) or Akt inhibitor (1 μm) for 45 min followed by attachment to collagen-coated plates as a function of time. FoxO3a and Actin expression levels were then measured. Un, untreated cells. Lower panel, fold-changes in FoxO3a expression (FoxO3a/actin) on collagen matrix in the presence or absence of PI3K or Akt inhibitor. The results are representative of three different experiments. E, left panel, serum-starved human lung fibroblasts were placed on fibronectin (FN)-, vitronectin (VN)-, or laminin (LN)-coated plates as a function of time and phosphorylated (p-Akt) and total Akt (Akt) levels were measured. Middle and right panels, cells were attached to collagen (100 μg/ml)-, fibronectin (10 μg/ml)-, vitronectin (10 μg/ml)-, or laminin (10 μg/ml)-coated plates as a function of time. Phosphorylated ERK (p-ERK, p-44/p-42) and ERK levels were then measured. F, human lung fibroblasts were preincubated with different doses of ERK inhibitor (ERKI) or Akt inhibitor (AktI, 1 μm) as indicated and phosphorylated FoxO3a (p-FoxO3a) and total FoxO3a (FoxO3a) levels were then measured. Numbers represent p-FoxO3a versus FoxO3a signal ratio.
    Figure Legend Snippet: FoxO3a activity decreases in response to fibroblast attachment to collagen. A, serum-starved human lung fibroblasts were attached to collagen-coated plates as a function of time and phosphorylated FoxO3a (p-FoxO3a, Ser-253) and actin levels were measured (upper panel). Cells were placed on fibronectin-, vitronectin-, and laminin-coated plates as a function of time and phosphorylated FoxO3a levels were measured (lower panel). Actin was used as a loading control. B, serum-starved human lung fibroblasts were attached at 0 to 100 μg/ml of collagen-coated plates for 30 min. FoxO3a and phosphorylated FoxO3a levels were then measured. Actin was used as a loading control. C, upper panel, human lung fibroblasts were serum-starved followed by preincubation with PI3K or Akt inhibitor as indicated for 45 min. Cells were then allowed to attach to collagen-coated plates for 30 min. FoxO3a expression levels were then measured. Actin was used as a loading control. PI3KI, PI3K inhibitor; AktI, Akt Inhibitor; s/s, serum-starved fibroblasts. DMSO, dimethyl sulfoxide control. Lower panel, fold-changes in FoxO3a expression (FoxO3a/actin) in the presence of PI3K or Akt inhibitor on collagen. The results are representative of three different experiments. D, upper panel, serum-starved lung fibroblasts were preincubated with PI3K (200 nm) or Akt inhibitor (1 μm) for 45 min followed by attachment to collagen-coated plates as a function of time. FoxO3a and Actin expression levels were then measured. Un, untreated cells. Lower panel, fold-changes in FoxO3a expression (FoxO3a/actin) on collagen matrix in the presence or absence of PI3K or Akt inhibitor. The results are representative of three different experiments. E, left panel, serum-starved human lung fibroblasts were placed on fibronectin (FN)-, vitronectin (VN)-, or laminin (LN)-coated plates as a function of time and phosphorylated (p-Akt) and total Akt (Akt) levels were measured. Middle and right panels, cells were attached to collagen (100 μg/ml)-, fibronectin (10 μg/ml)-, vitronectin (10 μg/ml)-, or laminin (10 μg/ml)-coated plates as a function of time. Phosphorylated ERK (p-ERK, p-44/p-42) and ERK levels were then measured. F, human lung fibroblasts were preincubated with different doses of ERK inhibitor (ERKI) or Akt inhibitor (AktI, 1 μm) as indicated and phosphorylated FoxO3a (p-FoxO3a) and total FoxO3a (FoxO3a) levels were then measured. Numbers represent p-FoxO3a versus FoxO3a signal ratio.

    Techniques Used: Activity Assay, Expressing

    β1-Integrin-collagen interaction suppresses the FoxO3a via PI3K/Akt pathway. A, human lung fibroblasts were preincubated with α1 to α5 and αv-integrin and/or β1-integrin blocking antibodies (1 μg/ml, respectively) for 45 min followed by the attachment to type I collagen for 30 min. FoxO3a levels were then measured. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. B, serum-starved human lung fibroblasts were preincubated with PI3K (0.2 μm) or Akt inhibitor (1 μm) for 45 min. Cells were then ligated with β1-integrin activating antibody (3 μg/ml) for 60 min. FoxO3a expression levels were measured. TS2/16, β1-integrin activating antibody; PI3KI, PI3K inhibitor; AktI, Akt inhibitor; IgG, isotype control. C, serum-starved human lung fibroblasts were preincubated with Akt inhibitor (1 μm) for 45 min. Cells were then stimulated with β1-integrin activating antibody TS2/16 (3 μg/ml) as a function of time. FoxO3a and actin expression levels were then measured. IgG, isotype control.
    Figure Legend Snippet: β1-Integrin-collagen interaction suppresses the FoxO3a via PI3K/Akt pathway. A, human lung fibroblasts were preincubated with α1 to α5 and αv-integrin and/or β1-integrin blocking antibodies (1 μg/ml, respectively) for 45 min followed by the attachment to type I collagen for 30 min. FoxO3a levels were then measured. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. B, serum-starved human lung fibroblasts were preincubated with PI3K (0.2 μm) or Akt inhibitor (1 μm) for 45 min. Cells were then ligated with β1-integrin activating antibody (3 μg/ml) for 60 min. FoxO3a expression levels were measured. TS2/16, β1-integrin activating antibody; PI3KI, PI3K inhibitor; AktI, Akt inhibitor; IgG, isotype control. C, serum-starved human lung fibroblasts were preincubated with Akt inhibitor (1 μm) for 45 min. Cells were then stimulated with β1-integrin activating antibody TS2/16 (3 μg/ml) as a function of time. FoxO3a and actin expression levels were then measured. IgG, isotype control.

    Techniques Used: Blocking Assay, Expressing

    Fibroblast adhesion to collagen decreases PTEN expression and activity. A, serum-starved fibroblasts were plated on dishes coated with type I collagen (100 μg/ml) and PTEN, FoxO3a, phosphorylated Akt (Ser-473), and actin levels were measured by Western analysis. B, serum-starved fibroblasts were plated on collagen for 15 and 60 min. p < 0.01 versus a reference value. Cells lysates were immunoprecipitated with anti-PTEN antibody, incubated with phosphatidylinositol (3,4,5)-trisphosphate substrate, and PTEN activity was quantified. To provide a reference value for PTEN activity under proliferation prohibitive conditions, PTEN activity was assessed in serum-starved fibroblasts cultured on tissue culture plastic plates under contact-inhibited conditions. C, upper panel, serum-starved fibroblasts were preincubated with α2- or β1-integrin blocking antibody (1 μg/ml), both (α2+β1), or isotype control antibody (IgG, 1 μg/ml) for 45 min and plated on collagen-coated plates for 30 min. PTEN levels were then measured. Actin was used as a loading control. Lower panel, shown are the phase-contrast microscopic cell morphologies at 30 min after plating the cells on collagen-coated plates. α2 and β1 represent α2 and β1 blocking antibody, respectively. IgG, isotype control. D, 5,000 cells were preincubated with integrin blocking antibodies for 45 min as indicated and the attached cells were counted at 30 min after plating the cells on collagen. E, serum-starved fibroblasts were pretreated with the indicated integrin blocking antibodies (1 μg/ml) and then plated on collagen-coated plates for 30 min. Total PTEN and actin levels were determined by Western analysis. As a reference, serum-starved (S/S) fibroblasts were trypsinized but not plated on collagen. F, PTEN expression was quantified by densitometry, normalized to actin.
    Figure Legend Snippet: Fibroblast adhesion to collagen decreases PTEN expression and activity. A, serum-starved fibroblasts were plated on dishes coated with type I collagen (100 μg/ml) and PTEN, FoxO3a, phosphorylated Akt (Ser-473), and actin levels were measured by Western analysis. B, serum-starved fibroblasts were plated on collagen for 15 and 60 min. p < 0.01 versus a reference value. Cells lysates were immunoprecipitated with anti-PTEN antibody, incubated with phosphatidylinositol (3,4,5)-trisphosphate substrate, and PTEN activity was quantified. To provide a reference value for PTEN activity under proliferation prohibitive conditions, PTEN activity was assessed in serum-starved fibroblasts cultured on tissue culture plastic plates under contact-inhibited conditions. C, upper panel, serum-starved fibroblasts were preincubated with α2- or β1-integrin blocking antibody (1 μg/ml), both (α2+β1), or isotype control antibody (IgG, 1 μg/ml) for 45 min and plated on collagen-coated plates for 30 min. PTEN levels were then measured. Actin was used as a loading control. Lower panel, shown are the phase-contrast microscopic cell morphologies at 30 min after plating the cells on collagen-coated plates. α2 and β1 represent α2 and β1 blocking antibody, respectively. IgG, isotype control. D, 5,000 cells were preincubated with integrin blocking antibodies for 45 min as indicated and the attached cells were counted at 30 min after plating the cells on collagen. E, serum-starved fibroblasts were pretreated with the indicated integrin blocking antibodies (1 μg/ml) and then plated on collagen-coated plates for 30 min. Total PTEN and actin levels were determined by Western analysis. As a reference, serum-starved (S/S) fibroblasts were trypsinized but not plated on collagen. F, PTEN expression was quantified by densitometry, normalized to actin.

    Techniques Used: Expressing, Activity Assay, Western Blot, Immunoprecipitation, Incubation, Cell Culture, Blocking Assay

    PTEN inhibition increases fibroblast proliferation via high Akt activity. A, upper panel, RT-PCR. Serum-starved fibroblasts were plated on collagen-coated dishes for the indicated times. The cells were lysed and total RNA was isolated by TRIzol. RT-PCR was performed as described under “Materials and Methods.” Lower panel, pulse-chase analysis. Fibroblasts were incubated in methionine-free medium followed by DMEM containing [35S]methionine as outlined under “Materials and Methods.” The cells were then plated on collagen-coated dishes in DMEM + 10% fetal calf serum for the indicated times. The cells were lysed, PTEN was immunoprecipitated from labeled cell extracts and resolved by SDS-PAGE. Radiolabeled PTEN was visualized by autoradiography. B, human lung fibroblasts were infected with adenovirus expressing wild type PTEN or mutant PTEN followed by serum starvation for 1 day. Cells were then attached to collagen-coated plates (100 μg/ml) for 48 h. Total Akt, phosphorylated Akt, and FoxO3a expression levels were then measured. Wt, wild type PTEN; Mu, phosphatase-dead mutant PTEN; Vec, empty vector. C, PTEN−/− and PTEN+/+ cells were serum-starved for 24 h followed by attachment to collagen-coated plates as a function of time. FoxO3a, Ser-253-phosphorylated FoxO3a, and Ser-473-phosphorylated Akt levels were measured. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. D, PTEN−/− fibroblasts infected with adenovirus expressing wild type PTEN (Wt), phosphatase-dead mutant PTEN (Mu), or empty vector (Vec) were attached to collagen-coated plates for 60 min. FoxO3a and Ser-253-phosphorylated FoxO3a expression levels were then measured. GAPDH was used as a loading control. E, 3,000 PTEN−/− and PTEN+/+ cells were incubated in DMEM and cell numbers were counted at 72 h as described under “Materials and Methods.” The results are representative of three different experiments. F, serum-starved lung fibroblasts infected with adenovirus expressing hyperactive Akt (Akt H) or dominant Akt (Akt DN, T308A, S473A) were seeded on collagen-coated plates for 30 min. FoxO3a, Ser-253 phosphorylated FoxO3a (p-FoxO3a), and Ser-473 phosphorylated Akt (p-Akt) levels were measured. GAPDH was used as a loading control.
    Figure Legend Snippet: PTEN inhibition increases fibroblast proliferation via high Akt activity. A, upper panel, RT-PCR. Serum-starved fibroblasts were plated on collagen-coated dishes for the indicated times. The cells were lysed and total RNA was isolated by TRIzol. RT-PCR was performed as described under “Materials and Methods.” Lower panel, pulse-chase analysis. Fibroblasts were incubated in methionine-free medium followed by DMEM containing [35S]methionine as outlined under “Materials and Methods.” The cells were then plated on collagen-coated dishes in DMEM + 10% fetal calf serum for the indicated times. The cells were lysed, PTEN was immunoprecipitated from labeled cell extracts and resolved by SDS-PAGE. Radiolabeled PTEN was visualized by autoradiography. B, human lung fibroblasts were infected with adenovirus expressing wild type PTEN or mutant PTEN followed by serum starvation for 1 day. Cells were then attached to collagen-coated plates (100 μg/ml) for 48 h. Total Akt, phosphorylated Akt, and FoxO3a expression levels were then measured. Wt, wild type PTEN; Mu, phosphatase-dead mutant PTEN; Vec, empty vector. C, PTEN−/− and PTEN+/+ cells were serum-starved for 24 h followed by attachment to collagen-coated plates as a function of time. FoxO3a, Ser-253-phosphorylated FoxO3a, and Ser-473-phosphorylated Akt levels were measured. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. D, PTEN−/− fibroblasts infected with adenovirus expressing wild type PTEN (Wt), phosphatase-dead mutant PTEN (Mu), or empty vector (Vec) were attached to collagen-coated plates for 60 min. FoxO3a and Ser-253-phosphorylated FoxO3a expression levels were then measured. GAPDH was used as a loading control. E, 3,000 PTEN−/− and PTEN+/+ cells were incubated in DMEM and cell numbers were counted at 72 h as described under “Materials and Methods.” The results are representative of three different experiments. F, serum-starved lung fibroblasts infected with adenovirus expressing hyperactive Akt (Akt H) or dominant Akt (Akt DN, T308A, S473A) were seeded on collagen-coated plates for 30 min. FoxO3a, Ser-253 phosphorylated FoxO3a (p-FoxO3a), and Ser-473 phosphorylated Akt (p-Akt) levels were measured. GAPDH was used as a loading control.

    Techniques Used: Inhibition, Activity Assay, Reverse Transcription Polymerase Chain Reaction, Isolation, Pulse Chase, Incubation, Immunoprecipitation, Labeling, SDS Page, Autoradiography, Infection, Expressing, Mutagenesis, Plasmid Preparation

    PP2A regulates FoxO3a on collagen matrix. A, serum-starved human lung fibroblasts were attached to collagen-coated plates (100 μg/ml) as a function of time and PP2A catalytic subunit expression levels were measured. Actin was used as a loading control. B, serum-starved lung fibroblasts were preincubated in the presence or absence of β1-integrin blocking antibody (1 μg/ml, BA) for 45 min. Cells were then attached to collagen-coated plates for 30 min. Western analysis was carried out to measure PP2A catalytic subunit expression (upper panel). PP2A activity was measured as described under “Materials and Methods” (lower panel). s/s, serum-starved cells; Un, untreated cells; BA, cells preincubated with β1-integrin blocking antibody; IgG, isotype control. C, lung fibroblasts infected with adenovirus expressing PP2A catalytic subunit or empty vector were serum-starved for 1 day. Cells were then attached to collagen-coated plates for 60 min. FoxO3a and HA-tagged proteins were then measured. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. PP2A, adenovirus expressing HA-tagged PP2A catalytic subunit; Vec, adenovirus expressing empty vector; Un, untreated cells; HA, HA-tagged antibody. D, serum-starved lung fibroblasts were preincubated with β1-integrin blocking antibody (1 μg/ml) in the presence or absence of 100 nm okadaic acid for 1 h. Cells were then attached to type I collagen for 30 min. FoxO3a expression levels were measured. GAPDH was used as a loading control. Un, untreated cells; P5D2, β1-integrin blocking antibody; DMSO, dimethyl sulfoxide control; OA, okadaic acid; IgG, isotype control. E, human lung fibroblasts were transfected with 10 nm PP2A siRNA (PP2A) or control siRNA (Control) along with untransfected (Un) cells followed by serum starvation for 1 day. Cells were then preincubated with β1-integrin blocking antibody (P5D2, 1 μg/ml) for 45 min and attached to collagen-coated plates for 30 min. FoxO3a and PP2A expression were then measured. Actin was used as a loading control.
    Figure Legend Snippet: PP2A regulates FoxO3a on collagen matrix. A, serum-starved human lung fibroblasts were attached to collagen-coated plates (100 μg/ml) as a function of time and PP2A catalytic subunit expression levels were measured. Actin was used as a loading control. B, serum-starved lung fibroblasts were preincubated in the presence or absence of β1-integrin blocking antibody (1 μg/ml, BA) for 45 min. Cells were then attached to collagen-coated plates for 30 min. Western analysis was carried out to measure PP2A catalytic subunit expression (upper panel). PP2A activity was measured as described under “Materials and Methods” (lower panel). s/s, serum-starved cells; Un, untreated cells; BA, cells preincubated with β1-integrin blocking antibody; IgG, isotype control. C, lung fibroblasts infected with adenovirus expressing PP2A catalytic subunit or empty vector were serum-starved for 1 day. Cells were then attached to collagen-coated plates for 60 min. FoxO3a and HA-tagged proteins were then measured. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. PP2A, adenovirus expressing HA-tagged PP2A catalytic subunit; Vec, adenovirus expressing empty vector; Un, untreated cells; HA, HA-tagged antibody. D, serum-starved lung fibroblasts were preincubated with β1-integrin blocking antibody (1 μg/ml) in the presence or absence of 100 nm okadaic acid for 1 h. Cells were then attached to type I collagen for 30 min. FoxO3a expression levels were measured. GAPDH was used as a loading control. Un, untreated cells; P5D2, β1-integrin blocking antibody; DMSO, dimethyl sulfoxide control; OA, okadaic acid; IgG, isotype control. E, human lung fibroblasts were transfected with 10 nm PP2A siRNA (PP2A) or control siRNA (Control) along with untransfected (Un) cells followed by serum starvation for 1 day. Cells were then preincubated with β1-integrin blocking antibody (P5D2, 1 μg/ml) for 45 min and attached to collagen-coated plates for 30 min. FoxO3a and PP2A expression were then measured. Actin was used as a loading control.

    Techniques Used: Expressing, Blocking Assay, Western Blot, Activity Assay, Infection, Plasmid Preparation, Transfection

    Cell attachment to collagen promotes caspase-3-dependent cleavage of FoxO3a. A, RT-PCR was carried out with primers designed for FoxO3a as described under “Materials and Methods.” Actin was used as a loading control. B, upper panel, serum-starved human lung fibroblasts were pretreated with caspase-3 inhibitor (30 nm) for 60 min. Cells were then attached to type I collagen as a function of time. FoxO3a levels were measured. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. DMSO, control. Lower panel, serum-starved human lung fibroblasts were pretreated with different doses of caspase-3 inhibitor (3 to 300 nm) for 60 min. Cells were then attached to type I collagen for 30 min. FoxO3a levels were then measured. GAPDH was used as a loading control. DMSO, control. C, serum-starved human lung fibroblasts were pretreated with β1-integrin blocking antibody (1 μg/ml) or isotype control antibody IgG (1 μg/ml) for 45 min. Cells were then attached to type I collagen (100 μg/ml) for 60 min. Caspase-3 activity assay was performed as described under “Materials and Methods.”
    Figure Legend Snippet: Cell attachment to collagen promotes caspase-3-dependent cleavage of FoxO3a. A, RT-PCR was carried out with primers designed for FoxO3a as described under “Materials and Methods.” Actin was used as a loading control. B, upper panel, serum-starved human lung fibroblasts were pretreated with caspase-3 inhibitor (30 nm) for 60 min. Cells were then attached to type I collagen as a function of time. FoxO3a levels were measured. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. DMSO, control. Lower panel, serum-starved human lung fibroblasts were pretreated with different doses of caspase-3 inhibitor (3 to 300 nm) for 60 min. Cells were then attached to type I collagen for 30 min. FoxO3a levels were then measured. GAPDH was used as a loading control. DMSO, control. C, serum-starved human lung fibroblasts were pretreated with β1-integrin blocking antibody (1 μg/ml) or isotype control antibody IgG (1 μg/ml) for 45 min. Cells were then attached to type I collagen (100 μg/ml) for 60 min. Caspase-3 activity assay was performed as described under “Materials and Methods.”

    Techniques Used: Cell Attachment Assay, Reverse Transcription Polymerase Chain Reaction, Blocking Assay, Caspase-3 Activity Assay

    FoxO3a increases p27 on the collagen matrix. A, serum-starved human lung fibroblasts were attached to collagen-coated plates as a function of time and p27 expression levels were measured. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. B, upper, FoxO3a siRNA (10 nm) transfected lung fibroblasts were serum-starved (s/s), and Western analysis was carried out to measure FoxO3a and p27 levels. GAPDH was used as a loading control. Lower, lung fibroblasts were ligated with β1-integrin activating antibody (TS2/16, 3 μg/ml) for 30 min in the presence or absence of FoxO3a siRNA. Western analysis was then carried out with antibodies as indicated. F3, FoxO3a siRNA; con, control siRNA; Un, untransfected cells. C, FoxO3a+/+ and Foxo3−/− cells were serum-starved for 1 day, and FoxO3a and p27 expression levels were measured using Western analysis. GAPDH was used as a loading control. D, FoxO3a+/+ cells were transfected with mouse FoxO3a siRNA (F3, 10 nm) or control siRNA (Con, 10 nm) and Western analysis was then carried out to measure FoxO3a and p27 expression levels. GAPDH was used as a loading control. Un, untransfected cells. E, serum-starved FoxO3a+/+ and FoxO3a−/− cells were attached to type I collagen for 30 min. p27 expression levels were then measured. GAPDH was used as a loading control. s/s, serum-starved fibroblasts. F, serum-starved human lung fibroblasts were preincubated with either β1-integrin blocking antibody (1 μg/ml) or isotype control mouse IgG (1 μg/ml) for 45 min. Cells were then attached to collagen-coated plates for 30 min. FoxO3a, PP2A, and p27 expression levels were then measured. GAPDH was used as a loading control. Un, untreated cells; BA, β1-integrin blocking antibody pretreated cells. G, serum-starved human lung fibroblasts were preincubated with β1-integrin blocking antibody (1 μg/ml) in the presence of 10, 100, or 200 nm or absence of okadaic acid for 60 min. Cells were then attached to type I collagen for 30 min. Western analysis was carried out to measure p27 expression levels. GAPDH was used as a loading control. s/s, serum-starved human lung fibroblasts; DMSO, control.
    Figure Legend Snippet: FoxO3a increases p27 on the collagen matrix. A, serum-starved human lung fibroblasts were attached to collagen-coated plates as a function of time and p27 expression levels were measured. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. B, upper, FoxO3a siRNA (10 nm) transfected lung fibroblasts were serum-starved (s/s), and Western analysis was carried out to measure FoxO3a and p27 levels. GAPDH was used as a loading control. Lower, lung fibroblasts were ligated with β1-integrin activating antibody (TS2/16, 3 μg/ml) for 30 min in the presence or absence of FoxO3a siRNA. Western analysis was then carried out with antibodies as indicated. F3, FoxO3a siRNA; con, control siRNA; Un, untransfected cells. C, FoxO3a+/+ and Foxo3−/− cells were serum-starved for 1 day, and FoxO3a and p27 expression levels were measured using Western analysis. GAPDH was used as a loading control. D, FoxO3a+/+ cells were transfected with mouse FoxO3a siRNA (F3, 10 nm) or control siRNA (Con, 10 nm) and Western analysis was then carried out to measure FoxO3a and p27 expression levels. GAPDH was used as a loading control. Un, untransfected cells. E, serum-starved FoxO3a+/+ and FoxO3a−/− cells were attached to type I collagen for 30 min. p27 expression levels were then measured. GAPDH was used as a loading control. s/s, serum-starved fibroblasts. F, serum-starved human lung fibroblasts were preincubated with either β1-integrin blocking antibody (1 μg/ml) or isotype control mouse IgG (1 μg/ml) for 45 min. Cells were then attached to collagen-coated plates for 30 min. FoxO3a, PP2A, and p27 expression levels were then measured. GAPDH was used as a loading control. Un, untreated cells; BA, β1-integrin blocking antibody pretreated cells. G, serum-starved human lung fibroblasts were preincubated with β1-integrin blocking antibody (1 μg/ml) in the presence of 10, 100, or 200 nm or absence of okadaic acid for 60 min. Cells were then attached to type I collagen for 30 min. Western analysis was carried out to measure p27 expression levels. GAPDH was used as a loading control. s/s, serum-starved human lung fibroblasts; DMSO, control.

    Techniques Used: Expressing, Transfection, Western Blot, Blocking Assay

    FoxO3a suppresses cell proliferation. A, FoxO3a siRNA or control siRNA-transfected human lung fibroblasts were grown in 96-well plates and an MTS assay was carried out as described under “Materials and Methods.” Note that FoxO3a siRNA-transfected fibroblasts had an enhanced proliferation rate compared with that of control siRNA or untransfected cells. *, p < 0.03 versus control siRNA. B, PTEN+/+ cells infected with adenovirus expressing dominant-negative FoxO3a (DN) or empty vector (Vec) were grown in 96-well plates and an MTS assay was carried out (upper panel). Dominant-negative FoxO3a expression was evaluated using Western analysis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control (middle panel). PTEN+/+ cells infected with adenovirus expressing dominant-negative FoxO3a or empty vector were incubated with DMEM and cells numbers were measured at 24 h (lower panel). *, p < 0.05 versus empty vector. C, 3 × 104 of FoxO3a+/+ and FoxO3a−/− cells were serum-starved for 1 day followed by attachment to type I collagen-coated plates for 24 h. Cells numbers were then measured. D, FoxO3a−/− cells were infected with adenovirus expressing wild type FoxO3a (FoxO3a) or empty vector (GFP) along with uninfected (Un) cells for 24 h. 1.2 × 104 of Foxo3a−/− fibroblasts were then plated on tissue culture plates in the presence of 0.5% serum for 72 h. Cell numbers were then measured using a Coulter counter. Upper, Western analysis of FoxO3a protein expression in FoxO3a−/− cells infected with adenovirus expressing FoxO3a wild type or empty vector or untransfected cells. GAPDH was used as a loading control. Lower, % change in cell numbers in FoxO3a−/− cells infected with adenovirus expressing FoxO3a wild type or empty vector or untransfected cells. *, p < 0.03 versus GFP empty vector.
    Figure Legend Snippet: FoxO3a suppresses cell proliferation. A, FoxO3a siRNA or control siRNA-transfected human lung fibroblasts were grown in 96-well plates and an MTS assay was carried out as described under “Materials and Methods.” Note that FoxO3a siRNA-transfected fibroblasts had an enhanced proliferation rate compared with that of control siRNA or untransfected cells. *, p < 0.03 versus control siRNA. B, PTEN+/+ cells infected with adenovirus expressing dominant-negative FoxO3a (DN) or empty vector (Vec) were grown in 96-well plates and an MTS assay was carried out (upper panel). Dominant-negative FoxO3a expression was evaluated using Western analysis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control (middle panel). PTEN+/+ cells infected with adenovirus expressing dominant-negative FoxO3a or empty vector were incubated with DMEM and cells numbers were measured at 24 h (lower panel). *, p < 0.05 versus empty vector. C, 3 × 104 of FoxO3a+/+ and FoxO3a−/− cells were serum-starved for 1 day followed by attachment to type I collagen-coated plates for 24 h. Cells numbers were then measured. D, FoxO3a−/− cells were infected with adenovirus expressing wild type FoxO3a (FoxO3a) or empty vector (GFP) along with uninfected (Un) cells for 24 h. 1.2 × 104 of Foxo3a−/− fibroblasts were then plated on tissue culture plates in the presence of 0.5% serum for 72 h. Cell numbers were then measured using a Coulter counter. Upper, Western analysis of FoxO3a protein expression in FoxO3a−/− cells infected with adenovirus expressing FoxO3a wild type or empty vector or untransfected cells. GAPDH was used as a loading control. Lower, % change in cell numbers in FoxO3a−/− cells infected with adenovirus expressing FoxO3a wild type or empty vector or untransfected cells. *, p < 0.03 versus GFP empty vector.

    Techniques Used: Transfection, MTS Assay, Infection, Expressing, Dominant Negative Mutation, Plasmid Preparation, Western Blot, Incubation

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    Cell Signaling Technology Inc foxo3a
    <t>FoxO3a</t> expression decreases on collagen. A, serum-starved human lung fibroblasts were attached to collagen (100 μg/ml, upper panel)-coated plates as a function of time. Akt and Ser-473-phosphorylated Akt levels were then measured. B, human lung fibroblasts were serum-starved for 2 days followed by attachment to collagen-coated plates as a function of time. FoxO3a, FoxO1, and FoxO4 expression levels were measured (upper and lower panels, respectively). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and actin were used as a loading control. C, serum-starved human lung fibroblasts were attached to fibronectin- (10 μg/ml), vitronectin- (10 μg/ml), and laminin (10 μg/ml)-coated plates as a function of time. FoxO3a expression levels were measured. Actin was used as a loading control.
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    1) Product Images from "?1-Integrin-Collagen Interaction Suppresses FoxO3a by the Coordination of Akt and PP2A"

    Article Title: ?1-Integrin-Collagen Interaction Suppresses FoxO3a by the Coordination of Akt and PP2A

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.052845

    FoxO3a expression decreases on collagen. A, serum-starved human lung fibroblasts were attached to collagen (100 μg/ml, upper panel)-coated plates as a function of time. Akt and Ser-473-phosphorylated Akt levels were then measured. B, human lung fibroblasts were serum-starved for 2 days followed by attachment to collagen-coated plates as a function of time. FoxO3a, FoxO1, and FoxO4 expression levels were measured (upper and lower panels, respectively). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and actin were used as a loading control. C, serum-starved human lung fibroblasts were attached to fibronectin- (10 μg/ml), vitronectin- (10 μg/ml), and laminin (10 μg/ml)-coated plates as a function of time. FoxO3a expression levels were measured. Actin was used as a loading control.
    Figure Legend Snippet: FoxO3a expression decreases on collagen. A, serum-starved human lung fibroblasts were attached to collagen (100 μg/ml, upper panel)-coated plates as a function of time. Akt and Ser-473-phosphorylated Akt levels were then measured. B, human lung fibroblasts were serum-starved for 2 days followed by attachment to collagen-coated plates as a function of time. FoxO3a, FoxO1, and FoxO4 expression levels were measured (upper and lower panels, respectively). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and actin were used as a loading control. C, serum-starved human lung fibroblasts were attached to fibronectin- (10 μg/ml), vitronectin- (10 μg/ml), and laminin (10 μg/ml)-coated plates as a function of time. FoxO3a expression levels were measured. Actin was used as a loading control.

    Techniques Used: Expressing

    FoxO3a activity decreases in response to fibroblast attachment to collagen. A, serum-starved human lung fibroblasts were attached to collagen-coated plates as a function of time and phosphorylated FoxO3a (p-FoxO3a, Ser-253) and actin levels were measured (upper panel). Cells were placed on fibronectin-, vitronectin-, and laminin-coated plates as a function of time and phosphorylated FoxO3a levels were measured (lower panel). Actin was used as a loading control. B, serum-starved human lung fibroblasts were attached at 0 to 100 μg/ml of collagen-coated plates for 30 min. FoxO3a and phosphorylated FoxO3a levels were then measured. Actin was used as a loading control. C, upper panel, human lung fibroblasts were serum-starved followed by preincubation with PI3K or Akt inhibitor as indicated for 45 min. Cells were then allowed to attach to collagen-coated plates for 30 min. FoxO3a expression levels were then measured. Actin was used as a loading control. PI3KI, PI3K inhibitor; AktI, Akt Inhibitor; s/s, serum-starved fibroblasts. DMSO, dimethyl sulfoxide control. Lower panel, fold-changes in FoxO3a expression (FoxO3a/actin) in the presence of PI3K or Akt inhibitor on collagen. The results are representative of three different experiments. D, upper panel, serum-starved lung fibroblasts were preincubated with PI3K (200 nm) or Akt inhibitor (1 μm) for 45 min followed by attachment to collagen-coated plates as a function of time. FoxO3a and Actin expression levels were then measured. Un, untreated cells. Lower panel, fold-changes in FoxO3a expression (FoxO3a/actin) on collagen matrix in the presence or absence of PI3K or Akt inhibitor. The results are representative of three different experiments. E, left panel, serum-starved human lung fibroblasts were placed on fibronectin (FN)-, vitronectin (VN)-, or laminin (LN)-coated plates as a function of time and phosphorylated (p-Akt) and total Akt (Akt) levels were measured. Middle and right panels, cells were attached to collagen (100 μg/ml)-, fibronectin (10 μg/ml)-, vitronectin (10 μg/ml)-, or laminin (10 μg/ml)-coated plates as a function of time. Phosphorylated ERK (p-ERK, p-44/p-42) and ERK levels were then measured. F, human lung fibroblasts were preincubated with different doses of ERK inhibitor (ERKI) or Akt inhibitor (AktI, 1 μm) as indicated and phosphorylated FoxO3a (p-FoxO3a) and total FoxO3a (FoxO3a) levels were then measured. Numbers represent p-FoxO3a versus FoxO3a signal ratio.
    Figure Legend Snippet: FoxO3a activity decreases in response to fibroblast attachment to collagen. A, serum-starved human lung fibroblasts were attached to collagen-coated plates as a function of time and phosphorylated FoxO3a (p-FoxO3a, Ser-253) and actin levels were measured (upper panel). Cells were placed on fibronectin-, vitronectin-, and laminin-coated plates as a function of time and phosphorylated FoxO3a levels were measured (lower panel). Actin was used as a loading control. B, serum-starved human lung fibroblasts were attached at 0 to 100 μg/ml of collagen-coated plates for 30 min. FoxO3a and phosphorylated FoxO3a levels were then measured. Actin was used as a loading control. C, upper panel, human lung fibroblasts were serum-starved followed by preincubation with PI3K or Akt inhibitor as indicated for 45 min. Cells were then allowed to attach to collagen-coated plates for 30 min. FoxO3a expression levels were then measured. Actin was used as a loading control. PI3KI, PI3K inhibitor; AktI, Akt Inhibitor; s/s, serum-starved fibroblasts. DMSO, dimethyl sulfoxide control. Lower panel, fold-changes in FoxO3a expression (FoxO3a/actin) in the presence of PI3K or Akt inhibitor on collagen. The results are representative of three different experiments. D, upper panel, serum-starved lung fibroblasts were preincubated with PI3K (200 nm) or Akt inhibitor (1 μm) for 45 min followed by attachment to collagen-coated plates as a function of time. FoxO3a and Actin expression levels were then measured. Un, untreated cells. Lower panel, fold-changes in FoxO3a expression (FoxO3a/actin) on collagen matrix in the presence or absence of PI3K or Akt inhibitor. The results are representative of three different experiments. E, left panel, serum-starved human lung fibroblasts were placed on fibronectin (FN)-, vitronectin (VN)-, or laminin (LN)-coated plates as a function of time and phosphorylated (p-Akt) and total Akt (Akt) levels were measured. Middle and right panels, cells were attached to collagen (100 μg/ml)-, fibronectin (10 μg/ml)-, vitronectin (10 μg/ml)-, or laminin (10 μg/ml)-coated plates as a function of time. Phosphorylated ERK (p-ERK, p-44/p-42) and ERK levels were then measured. F, human lung fibroblasts were preincubated with different doses of ERK inhibitor (ERKI) or Akt inhibitor (AktI, 1 μm) as indicated and phosphorylated FoxO3a (p-FoxO3a) and total FoxO3a (FoxO3a) levels were then measured. Numbers represent p-FoxO3a versus FoxO3a signal ratio.

    Techniques Used: Activity Assay, Expressing

    β1-Integrin-collagen interaction suppresses the FoxO3a via PI3K/Akt pathway. A, human lung fibroblasts were preincubated with α1 to α5 and αv-integrin and/or β1-integrin blocking antibodies (1 μg/ml, respectively) for 45 min followed by the attachment to type I collagen for 30 min. FoxO3a levels were then measured. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. B, serum-starved human lung fibroblasts were preincubated with PI3K (0.2 μm) or Akt inhibitor (1 μm) for 45 min. Cells were then ligated with β1-integrin activating antibody (3 μg/ml) for 60 min. FoxO3a expression levels were measured. TS2/16, β1-integrin activating antibody; PI3KI, PI3K inhibitor; AktI, Akt inhibitor; IgG, isotype control. C, serum-starved human lung fibroblasts were preincubated with Akt inhibitor (1 μm) for 45 min. Cells were then stimulated with β1-integrin activating antibody TS2/16 (3 μg/ml) as a function of time. FoxO3a and actin expression levels were then measured. IgG, isotype control.
    Figure Legend Snippet: β1-Integrin-collagen interaction suppresses the FoxO3a via PI3K/Akt pathway. A, human lung fibroblasts were preincubated with α1 to α5 and αv-integrin and/or β1-integrin blocking antibodies (1 μg/ml, respectively) for 45 min followed by the attachment to type I collagen for 30 min. FoxO3a levels were then measured. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. B, serum-starved human lung fibroblasts were preincubated with PI3K (0.2 μm) or Akt inhibitor (1 μm) for 45 min. Cells were then ligated with β1-integrin activating antibody (3 μg/ml) for 60 min. FoxO3a expression levels were measured. TS2/16, β1-integrin activating antibody; PI3KI, PI3K inhibitor; AktI, Akt inhibitor; IgG, isotype control. C, serum-starved human lung fibroblasts were preincubated with Akt inhibitor (1 μm) for 45 min. Cells were then stimulated with β1-integrin activating antibody TS2/16 (3 μg/ml) as a function of time. FoxO3a and actin expression levels were then measured. IgG, isotype control.

    Techniques Used: Blocking Assay, Expressing

    Fibroblast adhesion to collagen decreases PTEN expression and activity. A, serum-starved fibroblasts were plated on dishes coated with type I collagen (100 μg/ml) and PTEN, FoxO3a, phosphorylated Akt (Ser-473), and actin levels were measured by Western analysis. B, serum-starved fibroblasts were plated on collagen for 15 and 60 min. p < 0.01 versus a reference value. Cells lysates were immunoprecipitated with anti-PTEN antibody, incubated with phosphatidylinositol (3,4,5)-trisphosphate substrate, and PTEN activity was quantified. To provide a reference value for PTEN activity under proliferation prohibitive conditions, PTEN activity was assessed in serum-starved fibroblasts cultured on tissue culture plastic plates under contact-inhibited conditions. C, upper panel, serum-starved fibroblasts were preincubated with α2- or β1-integrin blocking antibody (1 μg/ml), both (α2+β1), or isotype control antibody (IgG, 1 μg/ml) for 45 min and plated on collagen-coated plates for 30 min. PTEN levels were then measured. Actin was used as a loading control. Lower panel, shown are the phase-contrast microscopic cell morphologies at 30 min after plating the cells on collagen-coated plates. α2 and β1 represent α2 and β1 blocking antibody, respectively. IgG, isotype control. D, 5,000 cells were preincubated with integrin blocking antibodies for 45 min as indicated and the attached cells were counted at 30 min after plating the cells on collagen. E, serum-starved fibroblasts were pretreated with the indicated integrin blocking antibodies (1 μg/ml) and then plated on collagen-coated plates for 30 min. Total PTEN and actin levels were determined by Western analysis. As a reference, serum-starved (S/S) fibroblasts were trypsinized but not plated on collagen. F, PTEN expression was quantified by densitometry, normalized to actin.
    Figure Legend Snippet: Fibroblast adhesion to collagen decreases PTEN expression and activity. A, serum-starved fibroblasts were plated on dishes coated with type I collagen (100 μg/ml) and PTEN, FoxO3a, phosphorylated Akt (Ser-473), and actin levels were measured by Western analysis. B, serum-starved fibroblasts were plated on collagen for 15 and 60 min. p < 0.01 versus a reference value. Cells lysates were immunoprecipitated with anti-PTEN antibody, incubated with phosphatidylinositol (3,4,5)-trisphosphate substrate, and PTEN activity was quantified. To provide a reference value for PTEN activity under proliferation prohibitive conditions, PTEN activity was assessed in serum-starved fibroblasts cultured on tissue culture plastic plates under contact-inhibited conditions. C, upper panel, serum-starved fibroblasts were preincubated with α2- or β1-integrin blocking antibody (1 μg/ml), both (α2+β1), or isotype control antibody (IgG, 1 μg/ml) for 45 min and plated on collagen-coated plates for 30 min. PTEN levels were then measured. Actin was used as a loading control. Lower panel, shown are the phase-contrast microscopic cell morphologies at 30 min after plating the cells on collagen-coated plates. α2 and β1 represent α2 and β1 blocking antibody, respectively. IgG, isotype control. D, 5,000 cells were preincubated with integrin blocking antibodies for 45 min as indicated and the attached cells were counted at 30 min after plating the cells on collagen. E, serum-starved fibroblasts were pretreated with the indicated integrin blocking antibodies (1 μg/ml) and then plated on collagen-coated plates for 30 min. Total PTEN and actin levels were determined by Western analysis. As a reference, serum-starved (S/S) fibroblasts were trypsinized but not plated on collagen. F, PTEN expression was quantified by densitometry, normalized to actin.

    Techniques Used: Expressing, Activity Assay, Western Blot, Immunoprecipitation, Incubation, Cell Culture, Blocking Assay

    PTEN inhibition increases fibroblast proliferation via high Akt activity. A, upper panel, RT-PCR. Serum-starved fibroblasts were plated on collagen-coated dishes for the indicated times. The cells were lysed and total RNA was isolated by TRIzol. RT-PCR was performed as described under “Materials and Methods.” Lower panel, pulse-chase analysis. Fibroblasts were incubated in methionine-free medium followed by DMEM containing [35S]methionine as outlined under “Materials and Methods.” The cells were then plated on collagen-coated dishes in DMEM + 10% fetal calf serum for the indicated times. The cells were lysed, PTEN was immunoprecipitated from labeled cell extracts and resolved by SDS-PAGE. Radiolabeled PTEN was visualized by autoradiography. B, human lung fibroblasts were infected with adenovirus expressing wild type PTEN or mutant PTEN followed by serum starvation for 1 day. Cells were then attached to collagen-coated plates (100 μg/ml) for 48 h. Total Akt, phosphorylated Akt, and FoxO3a expression levels were then measured. Wt, wild type PTEN; Mu, phosphatase-dead mutant PTEN; Vec, empty vector. C, PTEN−/− and PTEN+/+ cells were serum-starved for 24 h followed by attachment to collagen-coated plates as a function of time. FoxO3a, Ser-253-phosphorylated FoxO3a, and Ser-473-phosphorylated Akt levels were measured. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. D, PTEN−/− fibroblasts infected with adenovirus expressing wild type PTEN (Wt), phosphatase-dead mutant PTEN (Mu), or empty vector (Vec) were attached to collagen-coated plates for 60 min. FoxO3a and Ser-253-phosphorylated FoxO3a expression levels were then measured. GAPDH was used as a loading control. E, 3,000 PTEN−/− and PTEN+/+ cells were incubated in DMEM and cell numbers were counted at 72 h as described under “Materials and Methods.” The results are representative of three different experiments. F, serum-starved lung fibroblasts infected with adenovirus expressing hyperactive Akt (Akt H) or dominant Akt (Akt DN, T308A, S473A) were seeded on collagen-coated plates for 30 min. FoxO3a, Ser-253 phosphorylated FoxO3a (p-FoxO3a), and Ser-473 phosphorylated Akt (p-Akt) levels were measured. GAPDH was used as a loading control.
    Figure Legend Snippet: PTEN inhibition increases fibroblast proliferation via high Akt activity. A, upper panel, RT-PCR. Serum-starved fibroblasts were plated on collagen-coated dishes for the indicated times. The cells were lysed and total RNA was isolated by TRIzol. RT-PCR was performed as described under “Materials and Methods.” Lower panel, pulse-chase analysis. Fibroblasts were incubated in methionine-free medium followed by DMEM containing [35S]methionine as outlined under “Materials and Methods.” The cells were then plated on collagen-coated dishes in DMEM + 10% fetal calf serum for the indicated times. The cells were lysed, PTEN was immunoprecipitated from labeled cell extracts and resolved by SDS-PAGE. Radiolabeled PTEN was visualized by autoradiography. B, human lung fibroblasts were infected with adenovirus expressing wild type PTEN or mutant PTEN followed by serum starvation for 1 day. Cells were then attached to collagen-coated plates (100 μg/ml) for 48 h. Total Akt, phosphorylated Akt, and FoxO3a expression levels were then measured. Wt, wild type PTEN; Mu, phosphatase-dead mutant PTEN; Vec, empty vector. C, PTEN−/− and PTEN+/+ cells were serum-starved for 24 h followed by attachment to collagen-coated plates as a function of time. FoxO3a, Ser-253-phosphorylated FoxO3a, and Ser-473-phosphorylated Akt levels were measured. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. D, PTEN−/− fibroblasts infected with adenovirus expressing wild type PTEN (Wt), phosphatase-dead mutant PTEN (Mu), or empty vector (Vec) were attached to collagen-coated plates for 60 min. FoxO3a and Ser-253-phosphorylated FoxO3a expression levels were then measured. GAPDH was used as a loading control. E, 3,000 PTEN−/− and PTEN+/+ cells were incubated in DMEM and cell numbers were counted at 72 h as described under “Materials and Methods.” The results are representative of three different experiments. F, serum-starved lung fibroblasts infected with adenovirus expressing hyperactive Akt (Akt H) or dominant Akt (Akt DN, T308A, S473A) were seeded on collagen-coated plates for 30 min. FoxO3a, Ser-253 phosphorylated FoxO3a (p-FoxO3a), and Ser-473 phosphorylated Akt (p-Akt) levels were measured. GAPDH was used as a loading control.

    Techniques Used: Inhibition, Activity Assay, Reverse Transcription Polymerase Chain Reaction, Isolation, Pulse Chase, Incubation, Immunoprecipitation, Labeling, SDS Page, Autoradiography, Infection, Expressing, Mutagenesis, Plasmid Preparation

    PP2A regulates FoxO3a on collagen matrix. A, serum-starved human lung fibroblasts were attached to collagen-coated plates (100 μg/ml) as a function of time and PP2A catalytic subunit expression levels were measured. Actin was used as a loading control. B, serum-starved lung fibroblasts were preincubated in the presence or absence of β1-integrin blocking antibody (1 μg/ml, BA) for 45 min. Cells were then attached to collagen-coated plates for 30 min. Western analysis was carried out to measure PP2A catalytic subunit expression (upper panel). PP2A activity was measured as described under “Materials and Methods” (lower panel). s/s, serum-starved cells; Un, untreated cells; BA, cells preincubated with β1-integrin blocking antibody; IgG, isotype control. C, lung fibroblasts infected with adenovirus expressing PP2A catalytic subunit or empty vector were serum-starved for 1 day. Cells were then attached to collagen-coated plates for 60 min. FoxO3a and HA-tagged proteins were then measured. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. PP2A, adenovirus expressing HA-tagged PP2A catalytic subunit; Vec, adenovirus expressing empty vector; Un, untreated cells; HA, HA-tagged antibody. D, serum-starved lung fibroblasts were preincubated with β1-integrin blocking antibody (1 μg/ml) in the presence or absence of 100 nm okadaic acid for 1 h. Cells were then attached to type I collagen for 30 min. FoxO3a expression levels were measured. GAPDH was used as a loading control. Un, untreated cells; P5D2, β1-integrin blocking antibody; DMSO, dimethyl sulfoxide control; OA, okadaic acid; IgG, isotype control. E, human lung fibroblasts were transfected with 10 nm PP2A siRNA (PP2A) or control siRNA (Control) along with untransfected (Un) cells followed by serum starvation for 1 day. Cells were then preincubated with β1-integrin blocking antibody (P5D2, 1 μg/ml) for 45 min and attached to collagen-coated plates for 30 min. FoxO3a and PP2A expression were then measured. Actin was used as a loading control.
    Figure Legend Snippet: PP2A regulates FoxO3a on collagen matrix. A, serum-starved human lung fibroblasts were attached to collagen-coated plates (100 μg/ml) as a function of time and PP2A catalytic subunit expression levels were measured. Actin was used as a loading control. B, serum-starved lung fibroblasts were preincubated in the presence or absence of β1-integrin blocking antibody (1 μg/ml, BA) for 45 min. Cells were then attached to collagen-coated plates for 30 min. Western analysis was carried out to measure PP2A catalytic subunit expression (upper panel). PP2A activity was measured as described under “Materials and Methods” (lower panel). s/s, serum-starved cells; Un, untreated cells; BA, cells preincubated with β1-integrin blocking antibody; IgG, isotype control. C, lung fibroblasts infected with adenovirus expressing PP2A catalytic subunit or empty vector were serum-starved for 1 day. Cells were then attached to collagen-coated plates for 60 min. FoxO3a and HA-tagged proteins were then measured. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. PP2A, adenovirus expressing HA-tagged PP2A catalytic subunit; Vec, adenovirus expressing empty vector; Un, untreated cells; HA, HA-tagged antibody. D, serum-starved lung fibroblasts were preincubated with β1-integrin blocking antibody (1 μg/ml) in the presence or absence of 100 nm okadaic acid for 1 h. Cells were then attached to type I collagen for 30 min. FoxO3a expression levels were measured. GAPDH was used as a loading control. Un, untreated cells; P5D2, β1-integrin blocking antibody; DMSO, dimethyl sulfoxide control; OA, okadaic acid; IgG, isotype control. E, human lung fibroblasts were transfected with 10 nm PP2A siRNA (PP2A) or control siRNA (Control) along with untransfected (Un) cells followed by serum starvation for 1 day. Cells were then preincubated with β1-integrin blocking antibody (P5D2, 1 μg/ml) for 45 min and attached to collagen-coated plates for 30 min. FoxO3a and PP2A expression were then measured. Actin was used as a loading control.

    Techniques Used: Expressing, Blocking Assay, Western Blot, Activity Assay, Infection, Plasmid Preparation, Transfection

    Cell attachment to collagen promotes caspase-3-dependent cleavage of FoxO3a. A, RT-PCR was carried out with primers designed for FoxO3a as described under “Materials and Methods.” Actin was used as a loading control. B, upper panel, serum-starved human lung fibroblasts were pretreated with caspase-3 inhibitor (30 nm) for 60 min. Cells were then attached to type I collagen as a function of time. FoxO3a levels were measured. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. DMSO, control. Lower panel, serum-starved human lung fibroblasts were pretreated with different doses of caspase-3 inhibitor (3 to 300 nm) for 60 min. Cells were then attached to type I collagen for 30 min. FoxO3a levels were then measured. GAPDH was used as a loading control. DMSO, control. C, serum-starved human lung fibroblasts were pretreated with β1-integrin blocking antibody (1 μg/ml) or isotype control antibody IgG (1 μg/ml) for 45 min. Cells were then attached to type I collagen (100 μg/ml) for 60 min. Caspase-3 activity assay was performed as described under “Materials and Methods.”
    Figure Legend Snippet: Cell attachment to collagen promotes caspase-3-dependent cleavage of FoxO3a. A, RT-PCR was carried out with primers designed for FoxO3a as described under “Materials and Methods.” Actin was used as a loading control. B, upper panel, serum-starved human lung fibroblasts were pretreated with caspase-3 inhibitor (30 nm) for 60 min. Cells were then attached to type I collagen as a function of time. FoxO3a levels were measured. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. DMSO, control. Lower panel, serum-starved human lung fibroblasts were pretreated with different doses of caspase-3 inhibitor (3 to 300 nm) for 60 min. Cells were then attached to type I collagen for 30 min. FoxO3a levels were then measured. GAPDH was used as a loading control. DMSO, control. C, serum-starved human lung fibroblasts were pretreated with β1-integrin blocking antibody (1 μg/ml) or isotype control antibody IgG (1 μg/ml) for 45 min. Cells were then attached to type I collagen (100 μg/ml) for 60 min. Caspase-3 activity assay was performed as described under “Materials and Methods.”

    Techniques Used: Cell Attachment Assay, Reverse Transcription Polymerase Chain Reaction, Blocking Assay, Caspase-3 Activity Assay

    FoxO3a increases p27 on the collagen matrix. A, serum-starved human lung fibroblasts were attached to collagen-coated plates as a function of time and p27 expression levels were measured. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. B, upper, FoxO3a siRNA (10 nm) transfected lung fibroblasts were serum-starved (s/s), and Western analysis was carried out to measure FoxO3a and p27 levels. GAPDH was used as a loading control. Lower, lung fibroblasts were ligated with β1-integrin activating antibody (TS2/16, 3 μg/ml) for 30 min in the presence or absence of FoxO3a siRNA. Western analysis was then carried out with antibodies as indicated. F3, FoxO3a siRNA; con, control siRNA; Un, untransfected cells. C, FoxO3a+/+ and Foxo3−/− cells were serum-starved for 1 day, and FoxO3a and p27 expression levels were measured using Western analysis. GAPDH was used as a loading control. D, FoxO3a+/+ cells were transfected with mouse FoxO3a siRNA (F3, 10 nm) or control siRNA (Con, 10 nm) and Western analysis was then carried out to measure FoxO3a and p27 expression levels. GAPDH was used as a loading control. Un, untransfected cells. E, serum-starved FoxO3a+/+ and FoxO3a−/− cells were attached to type I collagen for 30 min. p27 expression levels were then measured. GAPDH was used as a loading control. s/s, serum-starved fibroblasts. F, serum-starved human lung fibroblasts were preincubated with either β1-integrin blocking antibody (1 μg/ml) or isotype control mouse IgG (1 μg/ml) for 45 min. Cells were then attached to collagen-coated plates for 30 min. FoxO3a, PP2A, and p27 expression levels were then measured. GAPDH was used as a loading control. Un, untreated cells; BA, β1-integrin blocking antibody pretreated cells. G, serum-starved human lung fibroblasts were preincubated with β1-integrin blocking antibody (1 μg/ml) in the presence of 10, 100, or 200 nm or absence of okadaic acid for 60 min. Cells were then attached to type I collagen for 30 min. Western analysis was carried out to measure p27 expression levels. GAPDH was used as a loading control. s/s, serum-starved human lung fibroblasts; DMSO, control.
    Figure Legend Snippet: FoxO3a increases p27 on the collagen matrix. A, serum-starved human lung fibroblasts were attached to collagen-coated plates as a function of time and p27 expression levels were measured. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. B, upper, FoxO3a siRNA (10 nm) transfected lung fibroblasts were serum-starved (s/s), and Western analysis was carried out to measure FoxO3a and p27 levels. GAPDH was used as a loading control. Lower, lung fibroblasts were ligated with β1-integrin activating antibody (TS2/16, 3 μg/ml) for 30 min in the presence or absence of FoxO3a siRNA. Western analysis was then carried out with antibodies as indicated. F3, FoxO3a siRNA; con, control siRNA; Un, untransfected cells. C, FoxO3a+/+ and Foxo3−/− cells were serum-starved for 1 day, and FoxO3a and p27 expression levels were measured using Western analysis. GAPDH was used as a loading control. D, FoxO3a+/+ cells were transfected with mouse FoxO3a siRNA (F3, 10 nm) or control siRNA (Con, 10 nm) and Western analysis was then carried out to measure FoxO3a and p27 expression levels. GAPDH was used as a loading control. Un, untransfected cells. E, serum-starved FoxO3a+/+ and FoxO3a−/− cells were attached to type I collagen for 30 min. p27 expression levels were then measured. GAPDH was used as a loading control. s/s, serum-starved fibroblasts. F, serum-starved human lung fibroblasts were preincubated with either β1-integrin blocking antibody (1 μg/ml) or isotype control mouse IgG (1 μg/ml) for 45 min. Cells were then attached to collagen-coated plates for 30 min. FoxO3a, PP2A, and p27 expression levels were then measured. GAPDH was used as a loading control. Un, untreated cells; BA, β1-integrin blocking antibody pretreated cells. G, serum-starved human lung fibroblasts were preincubated with β1-integrin blocking antibody (1 μg/ml) in the presence of 10, 100, or 200 nm or absence of okadaic acid for 60 min. Cells were then attached to type I collagen for 30 min. Western analysis was carried out to measure p27 expression levels. GAPDH was used as a loading control. s/s, serum-starved human lung fibroblasts; DMSO, control.

    Techniques Used: Expressing, Transfection, Western Blot, Blocking Assay

    FoxO3a suppresses cell proliferation. A, FoxO3a siRNA or control siRNA-transfected human lung fibroblasts were grown in 96-well plates and an MTS assay was carried out as described under “Materials and Methods.” Note that FoxO3a siRNA-transfected fibroblasts had an enhanced proliferation rate compared with that of control siRNA or untransfected cells. *, p < 0.03 versus control siRNA. B, PTEN+/+ cells infected with adenovirus expressing dominant-negative FoxO3a (DN) or empty vector (Vec) were grown in 96-well plates and an MTS assay was carried out (upper panel). Dominant-negative FoxO3a expression was evaluated using Western analysis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control (middle panel). PTEN+/+ cells infected with adenovirus expressing dominant-negative FoxO3a or empty vector were incubated with DMEM and cells numbers were measured at 24 h (lower panel). *, p < 0.05 versus empty vector. C, 3 × 104 of FoxO3a+/+ and FoxO3a−/− cells were serum-starved for 1 day followed by attachment to type I collagen-coated plates for 24 h. Cells numbers were then measured. D, FoxO3a−/− cells were infected with adenovirus expressing wild type FoxO3a (FoxO3a) or empty vector (GFP) along with uninfected (Un) cells for 24 h. 1.2 × 104 of Foxo3a−/− fibroblasts were then plated on tissue culture plates in the presence of 0.5% serum for 72 h. Cell numbers were then measured using a Coulter counter. Upper, Western analysis of FoxO3a protein expression in FoxO3a−/− cells infected with adenovirus expressing FoxO3a wild type or empty vector or untransfected cells. GAPDH was used as a loading control. Lower, % change in cell numbers in FoxO3a−/− cells infected with adenovirus expressing FoxO3a wild type or empty vector or untransfected cells. *, p < 0.03 versus GFP empty vector.
    Figure Legend Snippet: FoxO3a suppresses cell proliferation. A, FoxO3a siRNA or control siRNA-transfected human lung fibroblasts were grown in 96-well plates and an MTS assay was carried out as described under “Materials and Methods.” Note that FoxO3a siRNA-transfected fibroblasts had an enhanced proliferation rate compared with that of control siRNA or untransfected cells. *, p < 0.03 versus control siRNA. B, PTEN+/+ cells infected with adenovirus expressing dominant-negative FoxO3a (DN) or empty vector (Vec) were grown in 96-well plates and an MTS assay was carried out (upper panel). Dominant-negative FoxO3a expression was evaluated using Western analysis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control (middle panel). PTEN+/+ cells infected with adenovirus expressing dominant-negative FoxO3a or empty vector were incubated with DMEM and cells numbers were measured at 24 h (lower panel). *, p < 0.05 versus empty vector. C, 3 × 104 of FoxO3a+/+ and FoxO3a−/− cells were serum-starved for 1 day followed by attachment to type I collagen-coated plates for 24 h. Cells numbers were then measured. D, FoxO3a−/− cells were infected with adenovirus expressing wild type FoxO3a (FoxO3a) or empty vector (GFP) along with uninfected (Un) cells for 24 h. 1.2 × 104 of Foxo3a−/− fibroblasts were then plated on tissue culture plates in the presence of 0.5% serum for 72 h. Cell numbers were then measured using a Coulter counter. Upper, Western analysis of FoxO3a protein expression in FoxO3a−/− cells infected with adenovirus expressing FoxO3a wild type or empty vector or untransfected cells. GAPDH was used as a loading control. Lower, % change in cell numbers in FoxO3a−/− cells infected with adenovirus expressing FoxO3a wild type or empty vector or untransfected cells. *, p < 0.03 versus GFP empty vector.

    Techniques Used: Transfection, MTS Assay, Infection, Expressing, Dominant Negative Mutation, Plasmid Preparation, Western Blot, Incubation

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    Cell Signaling Technology Inc foxo3a
    <t>FoxO3a</t> expression decreases on collagen. A, serum-starved human lung fibroblasts were attached to collagen (100 μg/ml, upper panel)-coated plates as a function of time. Akt and Ser-473-phosphorylated Akt levels were then measured. B, human lung fibroblasts were serum-starved for 2 days followed by attachment to collagen-coated plates as a function of time. FoxO3a, FoxO1, and FoxO4 expression levels were measured (upper and lower panels, respectively). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and actin were used as a loading control. C, serum-starved human lung fibroblasts were attached to fibronectin- (10 μg/ml), vitronectin- (10 μg/ml), and laminin (10 μg/ml)-coated plates as a function of time. FoxO3a expression levels were measured. Actin was used as a loading control.
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    1) Product Images from "?1-Integrin-Collagen Interaction Suppresses FoxO3a by the Coordination of Akt and PP2A * "

    Article Title: ?1-Integrin-Collagen Interaction Suppresses FoxO3a by the Coordination of Akt and PP2A *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.052845

    FoxO3a expression decreases on collagen. A, serum-starved human lung fibroblasts were attached to collagen (100 μg/ml, upper panel)-coated plates as a function of time. Akt and Ser-473-phosphorylated Akt levels were then measured. B, human lung fibroblasts were serum-starved for 2 days followed by attachment to collagen-coated plates as a function of time. FoxO3a, FoxO1, and FoxO4 expression levels were measured (upper and lower panels, respectively). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and actin were used as a loading control. C, serum-starved human lung fibroblasts were attached to fibronectin- (10 μg/ml), vitronectin- (10 μg/ml), and laminin (10 μg/ml)-coated plates as a function of time. FoxO3a expression levels were measured. Actin was used as a loading control.
    Figure Legend Snippet: FoxO3a expression decreases on collagen. A, serum-starved human lung fibroblasts were attached to collagen (100 μg/ml, upper panel)-coated plates as a function of time. Akt and Ser-473-phosphorylated Akt levels were then measured. B, human lung fibroblasts were serum-starved for 2 days followed by attachment to collagen-coated plates as a function of time. FoxO3a, FoxO1, and FoxO4 expression levels were measured (upper and lower panels, respectively). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and actin were used as a loading control. C, serum-starved human lung fibroblasts were attached to fibronectin- (10 μg/ml), vitronectin- (10 μg/ml), and laminin (10 μg/ml)-coated plates as a function of time. FoxO3a expression levels were measured. Actin was used as a loading control.

    Techniques Used: Expressing

    FoxO3a activity decreases in response to fibroblast attachment to collagen. A, serum-starved human lung fibroblasts were attached to collagen-coated plates as a function of time and phosphorylated FoxO3a (p-FoxO3a, Ser-253) and actin levels were measured (upper panel). Cells were placed on fibronectin-, vitronectin-, and laminin-coated plates as a function of time and phosphorylated FoxO3a levels were measured (lower panel). Actin was used as a loading control. B, serum-starved human lung fibroblasts were attached at 0 to 100 μg/ml of collagen-coated plates for 30 min. FoxO3a and phosphorylated FoxO3a levels were then measured. Actin was used as a loading control. C, upper panel, human lung fibroblasts were serum-starved followed by preincubation with PI3K or Akt inhibitor as indicated for 45 min. Cells were then allowed to attach to collagen-coated plates for 30 min. FoxO3a expression levels were then measured. Actin was used as a loading control. PI3KI, PI3K inhibitor; AktI, Akt Inhibitor; s/s, serum-starved fibroblasts. DMSO, dimethyl sulfoxide control. Lower panel, fold-changes in FoxO3a expression (FoxO3a/actin) in the presence of PI3K or Akt inhibitor on collagen. The results are representative of three different experiments. D, upper panel, serum-starved lung fibroblasts were preincubated with PI3K (200 nm) or Akt inhibitor (1 μm) for 45 min followed by attachment to collagen-coated plates as a function of time. FoxO3a and Actin expression levels were then measured. Un, untreated cells. Lower panel, fold-changes in FoxO3a expression (FoxO3a/actin) on collagen matrix in the presence or absence of PI3K or Akt inhibitor. The results are representative of three different experiments. E, left panel, serum-starved human lung fibroblasts were placed on fibronectin (FN)-, vitronectin (VN)-, or laminin (LN)-coated plates as a function of time and phosphorylated (p-Akt) and total Akt (Akt) levels were measured. Middle and right panels, cells were attached to collagen (100 μg/ml)-, fibronectin (10 μg/ml)-, vitronectin (10 μg/ml)-, or laminin (10 μg/ml)-coated plates as a function of time. Phosphorylated ERK (p-ERK, p-44/p-42) and ERK levels were then measured. F, human lung fibroblasts were preincubated with different doses of ERK inhibitor (ERKI) or Akt inhibitor (AktI, 1 μm) as indicated and phosphorylated FoxO3a (p-FoxO3a) and total FoxO3a (FoxO3a) levels were then measured. Numbers represent p-FoxO3a versus FoxO3a signal ratio.
    Figure Legend Snippet: FoxO3a activity decreases in response to fibroblast attachment to collagen. A, serum-starved human lung fibroblasts were attached to collagen-coated plates as a function of time and phosphorylated FoxO3a (p-FoxO3a, Ser-253) and actin levels were measured (upper panel). Cells were placed on fibronectin-, vitronectin-, and laminin-coated plates as a function of time and phosphorylated FoxO3a levels were measured (lower panel). Actin was used as a loading control. B, serum-starved human lung fibroblasts were attached at 0 to 100 μg/ml of collagen-coated plates for 30 min. FoxO3a and phosphorylated FoxO3a levels were then measured. Actin was used as a loading control. C, upper panel, human lung fibroblasts were serum-starved followed by preincubation with PI3K or Akt inhibitor as indicated for 45 min. Cells were then allowed to attach to collagen-coated plates for 30 min. FoxO3a expression levels were then measured. Actin was used as a loading control. PI3KI, PI3K inhibitor; AktI, Akt Inhibitor; s/s, serum-starved fibroblasts. DMSO, dimethyl sulfoxide control. Lower panel, fold-changes in FoxO3a expression (FoxO3a/actin) in the presence of PI3K or Akt inhibitor on collagen. The results are representative of three different experiments. D, upper panel, serum-starved lung fibroblasts were preincubated with PI3K (200 nm) or Akt inhibitor (1 μm) for 45 min followed by attachment to collagen-coated plates as a function of time. FoxO3a and Actin expression levels were then measured. Un, untreated cells. Lower panel, fold-changes in FoxO3a expression (FoxO3a/actin) on collagen matrix in the presence or absence of PI3K or Akt inhibitor. The results are representative of three different experiments. E, left panel, serum-starved human lung fibroblasts were placed on fibronectin (FN)-, vitronectin (VN)-, or laminin (LN)-coated plates as a function of time and phosphorylated (p-Akt) and total Akt (Akt) levels were measured. Middle and right panels, cells were attached to collagen (100 μg/ml)-, fibronectin (10 μg/ml)-, vitronectin (10 μg/ml)-, or laminin (10 μg/ml)-coated plates as a function of time. Phosphorylated ERK (p-ERK, p-44/p-42) and ERK levels were then measured. F, human lung fibroblasts were preincubated with different doses of ERK inhibitor (ERKI) or Akt inhibitor (AktI, 1 μm) as indicated and phosphorylated FoxO3a (p-FoxO3a) and total FoxO3a (FoxO3a) levels were then measured. Numbers represent p-FoxO3a versus FoxO3a signal ratio.

    Techniques Used: Activity Assay, Expressing

    β1-Integrin-collagen interaction suppresses the FoxO3a via PI3K/Akt pathway. A, human lung fibroblasts were preincubated with α1 to α5 and αv-integrin and/or β1-integrin blocking antibodies (1 μg/ml, respectively) for 45 min followed by the attachment to type I collagen for 30 min. FoxO3a levels were then measured. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. B, serum-starved human lung fibroblasts were preincubated with PI3K (0.2 μm) or Akt inhibitor (1 μm) for 45 min. Cells were then ligated with β1-integrin activating antibody (3 μg/ml) for 60 min. FoxO3a expression levels were measured. TS2/16, β1-integrin activating antibody; PI3KI, PI3K inhibitor; AktI, Akt inhibitor; IgG, isotype control. C, serum-starved human lung fibroblasts were preincubated with Akt inhibitor (1 μm) for 45 min. Cells were then stimulated with β1-integrin activating antibody TS2/16 (3 μg/ml) as a function of time. FoxO3a and actin expression levels were then measured. IgG, isotype control.
    Figure Legend Snippet: β1-Integrin-collagen interaction suppresses the FoxO3a via PI3K/Akt pathway. A, human lung fibroblasts were preincubated with α1 to α5 and αv-integrin and/or β1-integrin blocking antibodies (1 μg/ml, respectively) for 45 min followed by the attachment to type I collagen for 30 min. FoxO3a levels were then measured. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. B, serum-starved human lung fibroblasts were preincubated with PI3K (0.2 μm) or Akt inhibitor (1 μm) for 45 min. Cells were then ligated with β1-integrin activating antibody (3 μg/ml) for 60 min. FoxO3a expression levels were measured. TS2/16, β1-integrin activating antibody; PI3KI, PI3K inhibitor; AktI, Akt inhibitor; IgG, isotype control. C, serum-starved human lung fibroblasts were preincubated with Akt inhibitor (1 μm) for 45 min. Cells were then stimulated with β1-integrin activating antibody TS2/16 (3 μg/ml) as a function of time. FoxO3a and actin expression levels were then measured. IgG, isotype control.

    Techniques Used: Blocking Assay, Expressing

    Fibroblast adhesion to collagen decreases PTEN expression and activity. A, serum-starved fibroblasts were plated on dishes coated with type I collagen (100 μg/ml) and PTEN, FoxO3a, phosphorylated Akt (Ser-473), and actin levels were measured by Western analysis. B, serum-starved fibroblasts were plated on collagen for 15 and 60 min. p < 0.01 versus a reference value. Cells lysates were immunoprecipitated with anti-PTEN antibody, incubated with phosphatidylinositol (3,4,5)-trisphosphate substrate, and PTEN activity was quantified. To provide a reference value for PTEN activity under proliferation prohibitive conditions, PTEN activity was assessed in serum-starved fibroblasts cultured on tissue culture plastic plates under contact-inhibited conditions. C, upper panel, serum-starved fibroblasts were preincubated with α2- or β1-integrin blocking antibody (1 μg/ml), both (α2+β1), or isotype control antibody (IgG, 1 μg/ml) for 45 min and plated on collagen-coated plates for 30 min. PTEN levels were then measured. Actin was used as a loading control. Lower panel, shown are the phase-contrast microscopic cell morphologies at 30 min after plating the cells on collagen-coated plates. α2 and β1 represent α2 and β1 blocking antibody, respectively. IgG, isotype control. D, 5,000 cells were preincubated with integrin blocking antibodies for 45 min as indicated and the attached cells were counted at 30 min after plating the cells on collagen. E, serum-starved fibroblasts were pretreated with the indicated integrin blocking antibodies (1 μg/ml) and then plated on collagen-coated plates for 30 min. Total PTEN and actin levels were determined by Western analysis. As a reference, serum-starved (S/S) fibroblasts were trypsinized but not plated on collagen. F, PTEN expression was quantified by densitometry, normalized to actin.
    Figure Legend Snippet: Fibroblast adhesion to collagen decreases PTEN expression and activity. A, serum-starved fibroblasts were plated on dishes coated with type I collagen (100 μg/ml) and PTEN, FoxO3a, phosphorylated Akt (Ser-473), and actin levels were measured by Western analysis. B, serum-starved fibroblasts were plated on collagen for 15 and 60 min. p < 0.01 versus a reference value. Cells lysates were immunoprecipitated with anti-PTEN antibody, incubated with phosphatidylinositol (3,4,5)-trisphosphate substrate, and PTEN activity was quantified. To provide a reference value for PTEN activity under proliferation prohibitive conditions, PTEN activity was assessed in serum-starved fibroblasts cultured on tissue culture plastic plates under contact-inhibited conditions. C, upper panel, serum-starved fibroblasts were preincubated with α2- or β1-integrin blocking antibody (1 μg/ml), both (α2+β1), or isotype control antibody (IgG, 1 μg/ml) for 45 min and plated on collagen-coated plates for 30 min. PTEN levels were then measured. Actin was used as a loading control. Lower panel, shown are the phase-contrast microscopic cell morphologies at 30 min after plating the cells on collagen-coated plates. α2 and β1 represent α2 and β1 blocking antibody, respectively. IgG, isotype control. D, 5,000 cells were preincubated with integrin blocking antibodies for 45 min as indicated and the attached cells were counted at 30 min after plating the cells on collagen. E, serum-starved fibroblasts were pretreated with the indicated integrin blocking antibodies (1 μg/ml) and then plated on collagen-coated plates for 30 min. Total PTEN and actin levels were determined by Western analysis. As a reference, serum-starved (S/S) fibroblasts were trypsinized but not plated on collagen. F, PTEN expression was quantified by densitometry, normalized to actin.

    Techniques Used: Expressing, Activity Assay, Western Blot, Immunoprecipitation, Incubation, Cell Culture, Blocking Assay

    PTEN inhibition increases fibroblast proliferation via high Akt activity. A, upper panel, RT-PCR. Serum-starved fibroblasts were plated on collagen-coated dishes for the indicated times. The cells were lysed and total RNA was isolated by TRIzol. RT-PCR was performed as described under “Materials and Methods.” Lower panel, pulse-chase analysis. Fibroblasts were incubated in methionine-free medium followed by DMEM containing [35S]methionine as outlined under “Materials and Methods.” The cells were then plated on collagen-coated dishes in DMEM + 10% fetal calf serum for the indicated times. The cells were lysed, PTEN was immunoprecipitated from labeled cell extracts and resolved by SDS-PAGE. Radiolabeled PTEN was visualized by autoradiography. B, human lung fibroblasts were infected with adenovirus expressing wild type PTEN or mutant PTEN followed by serum starvation for 1 day. Cells were then attached to collagen-coated plates (100 μg/ml) for 48 h. Total Akt, phosphorylated Akt, and FoxO3a expression levels were then measured. Wt, wild type PTEN; Mu, phosphatase-dead mutant PTEN; Vec, empty vector. C, PTEN−/− and PTEN+/+ cells were serum-starved for 24 h followed by attachment to collagen-coated plates as a function of time. FoxO3a, Ser-253-phosphorylated FoxO3a, and Ser-473-phosphorylated Akt levels were measured. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. D, PTEN−/− fibroblasts infected with adenovirus expressing wild type PTEN (Wt), phosphatase-dead mutant PTEN (Mu), or empty vector (Vec) were attached to collagen-coated plates for 60 min. FoxO3a and Ser-253-phosphorylated FoxO3a expression levels were then measured. GAPDH was used as a loading control. E, 3,000 PTEN−/− and PTEN+/+ cells were incubated in DMEM and cell numbers were counted at 72 h as described under “Materials and Methods.” The results are representative of three different experiments. F, serum-starved lung fibroblasts infected with adenovirus expressing hyperactive Akt (Akt H) or dominant Akt (Akt DN, T308A, S473A) were seeded on collagen-coated plates for 30 min. FoxO3a, Ser-253 phosphorylated FoxO3a (p-FoxO3a), and Ser-473 phosphorylated Akt (p-Akt) levels were measured. GAPDH was used as a loading control.
    Figure Legend Snippet: PTEN inhibition increases fibroblast proliferation via high Akt activity. A, upper panel, RT-PCR. Serum-starved fibroblasts were plated on collagen-coated dishes for the indicated times. The cells were lysed and total RNA was isolated by TRIzol. RT-PCR was performed as described under “Materials and Methods.” Lower panel, pulse-chase analysis. Fibroblasts were incubated in methionine-free medium followed by DMEM containing [35S]methionine as outlined under “Materials and Methods.” The cells were then plated on collagen-coated dishes in DMEM + 10% fetal calf serum for the indicated times. The cells were lysed, PTEN was immunoprecipitated from labeled cell extracts and resolved by SDS-PAGE. Radiolabeled PTEN was visualized by autoradiography. B, human lung fibroblasts were infected with adenovirus expressing wild type PTEN or mutant PTEN followed by serum starvation for 1 day. Cells were then attached to collagen-coated plates (100 μg/ml) for 48 h. Total Akt, phosphorylated Akt, and FoxO3a expression levels were then measured. Wt, wild type PTEN; Mu, phosphatase-dead mutant PTEN; Vec, empty vector. C, PTEN−/− and PTEN+/+ cells were serum-starved for 24 h followed by attachment to collagen-coated plates as a function of time. FoxO3a, Ser-253-phosphorylated FoxO3a, and Ser-473-phosphorylated Akt levels were measured. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. D, PTEN−/− fibroblasts infected with adenovirus expressing wild type PTEN (Wt), phosphatase-dead mutant PTEN (Mu), or empty vector (Vec) were attached to collagen-coated plates for 60 min. FoxO3a and Ser-253-phosphorylated FoxO3a expression levels were then measured. GAPDH was used as a loading control. E, 3,000 PTEN−/− and PTEN+/+ cells were incubated in DMEM and cell numbers were counted at 72 h as described under “Materials and Methods.” The results are representative of three different experiments. F, serum-starved lung fibroblasts infected with adenovirus expressing hyperactive Akt (Akt H) or dominant Akt (Akt DN, T308A, S473A) were seeded on collagen-coated plates for 30 min. FoxO3a, Ser-253 phosphorylated FoxO3a (p-FoxO3a), and Ser-473 phosphorylated Akt (p-Akt) levels were measured. GAPDH was used as a loading control.

    Techniques Used: Inhibition, Activity Assay, Reverse Transcription Polymerase Chain Reaction, Isolation, Pulse Chase, Incubation, Immunoprecipitation, Labeling, SDS Page, Autoradiography, Infection, Expressing, Mutagenesis, Plasmid Preparation

    PP2A regulates FoxO3a on collagen matrix. A, serum-starved human lung fibroblasts were attached to collagen-coated plates (100 μg/ml) as a function of time and PP2A catalytic subunit expression levels were measured. Actin was used as a loading control. B, serum-starved lung fibroblasts were preincubated in the presence or absence of β1-integrin blocking antibody (1 μg/ml, BA) for 45 min. Cells were then attached to collagen-coated plates for 30 min. Western analysis was carried out to measure PP2A catalytic subunit expression (upper panel). PP2A activity was measured as described under “Materials and Methods” (lower panel). s/s, serum-starved cells; Un, untreated cells; BA, cells preincubated with β1-integrin blocking antibody; IgG, isotype control. C, lung fibroblasts infected with adenovirus expressing PP2A catalytic subunit or empty vector were serum-starved for 1 day. Cells were then attached to collagen-coated plates for 60 min. FoxO3a and HA-tagged proteins were then measured. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. PP2A, adenovirus expressing HA-tagged PP2A catalytic subunit; Vec, adenovirus expressing empty vector; Un, untreated cells; HA, HA-tagged antibody. D, serum-starved lung fibroblasts were preincubated with β1-integrin blocking antibody (1 μg/ml) in the presence or absence of 100 nm okadaic acid for 1 h. Cells were then attached to type I collagen for 30 min. FoxO3a expression levels were measured. GAPDH was used as a loading control. Un, untreated cells; P5D2, β1-integrin blocking antibody; DMSO, dimethyl sulfoxide control; OA, okadaic acid; IgG, isotype control. E, human lung fibroblasts were transfected with 10 nm PP2A siRNA (PP2A) or control siRNA (Control) along with untransfected (Un) cells followed by serum starvation for 1 day. Cells were then preincubated with β1-integrin blocking antibody (P5D2, 1 μg/ml) for 45 min and attached to collagen-coated plates for 30 min. FoxO3a and PP2A expression were then measured. Actin was used as a loading control.
    Figure Legend Snippet: PP2A regulates FoxO3a on collagen matrix. A, serum-starved human lung fibroblasts were attached to collagen-coated plates (100 μg/ml) as a function of time and PP2A catalytic subunit expression levels were measured. Actin was used as a loading control. B, serum-starved lung fibroblasts were preincubated in the presence or absence of β1-integrin blocking antibody (1 μg/ml, BA) for 45 min. Cells were then attached to collagen-coated plates for 30 min. Western analysis was carried out to measure PP2A catalytic subunit expression (upper panel). PP2A activity was measured as described under “Materials and Methods” (lower panel). s/s, serum-starved cells; Un, untreated cells; BA, cells preincubated with β1-integrin blocking antibody; IgG, isotype control. C, lung fibroblasts infected with adenovirus expressing PP2A catalytic subunit or empty vector were serum-starved for 1 day. Cells were then attached to collagen-coated plates for 60 min. FoxO3a and HA-tagged proteins were then measured. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. PP2A, adenovirus expressing HA-tagged PP2A catalytic subunit; Vec, adenovirus expressing empty vector; Un, untreated cells; HA, HA-tagged antibody. D, serum-starved lung fibroblasts were preincubated with β1-integrin blocking antibody (1 μg/ml) in the presence or absence of 100 nm okadaic acid for 1 h. Cells were then attached to type I collagen for 30 min. FoxO3a expression levels were measured. GAPDH was used as a loading control. Un, untreated cells; P5D2, β1-integrin blocking antibody; DMSO, dimethyl sulfoxide control; OA, okadaic acid; IgG, isotype control. E, human lung fibroblasts were transfected with 10 nm PP2A siRNA (PP2A) or control siRNA (Control) along with untransfected (Un) cells followed by serum starvation for 1 day. Cells were then preincubated with β1-integrin blocking antibody (P5D2, 1 μg/ml) for 45 min and attached to collagen-coated plates for 30 min. FoxO3a and PP2A expression were then measured. Actin was used as a loading control.

    Techniques Used: Expressing, Blocking Assay, Western Blot, Activity Assay, Infection, Plasmid Preparation, Transfection

    Cell attachment to collagen promotes caspase-3-dependent cleavage of FoxO3a. A, RT-PCR was carried out with primers designed for FoxO3a as described under “Materials and Methods.” Actin was used as a loading control. B, upper panel, serum-starved human lung fibroblasts were pretreated with caspase-3 inhibitor (30 nm) for 60 min. Cells were then attached to type I collagen as a function of time. FoxO3a levels were measured. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. DMSO, control. Lower panel, serum-starved human lung fibroblasts were pretreated with different doses of caspase-3 inhibitor (3 to 300 nm) for 60 min. Cells were then attached to type I collagen for 30 min. FoxO3a levels were then measured. GAPDH was used as a loading control. DMSO, control. C, serum-starved human lung fibroblasts were pretreated with β1-integrin blocking antibody (1 μg/ml) or isotype control antibody IgG (1 μg/ml) for 45 min. Cells were then attached to type I collagen (100 μg/ml) for 60 min. Caspase-3 activity assay was performed as described under “Materials and Methods.”
    Figure Legend Snippet: Cell attachment to collagen promotes caspase-3-dependent cleavage of FoxO3a. A, RT-PCR was carried out with primers designed for FoxO3a as described under “Materials and Methods.” Actin was used as a loading control. B, upper panel, serum-starved human lung fibroblasts were pretreated with caspase-3 inhibitor (30 nm) for 60 min. Cells were then attached to type I collagen as a function of time. FoxO3a levels were measured. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. DMSO, control. Lower panel, serum-starved human lung fibroblasts were pretreated with different doses of caspase-3 inhibitor (3 to 300 nm) for 60 min. Cells were then attached to type I collagen for 30 min. FoxO3a levels were then measured. GAPDH was used as a loading control. DMSO, control. C, serum-starved human lung fibroblasts were pretreated with β1-integrin blocking antibody (1 μg/ml) or isotype control antibody IgG (1 μg/ml) for 45 min. Cells were then attached to type I collagen (100 μg/ml) for 60 min. Caspase-3 activity assay was performed as described under “Materials and Methods.”

    Techniques Used: Cell Attachment Assay, Reverse Transcription Polymerase Chain Reaction, Blocking Assay, Caspase-3 Activity Assay

    FoxO3a increases p27 on the collagen matrix. A, serum-starved human lung fibroblasts were attached to collagen-coated plates as a function of time and p27 expression levels were measured. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. B, upper, FoxO3a siRNA (10 nm) transfected lung fibroblasts were serum-starved (s/s), and Western analysis was carried out to measure FoxO3a and p27 levels. GAPDH was used as a loading control. Lower, lung fibroblasts were ligated with β1-integrin activating antibody (TS2/16, 3 μg/ml) for 30 min in the presence or absence of FoxO3a siRNA. Western analysis was then carried out with antibodies as indicated. F3, FoxO3a siRNA; con, control siRNA; Un, untransfected cells. C, FoxO3a+/+ and Foxo3−/− cells were serum-starved for 1 day, and FoxO3a and p27 expression levels were measured using Western analysis. GAPDH was used as a loading control. D, FoxO3a+/+ cells were transfected with mouse FoxO3a siRNA (F3, 10 nm) or control siRNA (Con, 10 nm) and Western analysis was then carried out to measure FoxO3a and p27 expression levels. GAPDH was used as a loading control. Un, untransfected cells. E, serum-starved FoxO3a+/+ and FoxO3a−/− cells were attached to type I collagen for 30 min. p27 expression levels were then measured. GAPDH was used as a loading control. s/s, serum-starved fibroblasts. F, serum-starved human lung fibroblasts were preincubated with either β1-integrin blocking antibody (1 μg/ml) or isotype control mouse IgG (1 μg/ml) for 45 min. Cells were then attached to collagen-coated plates for 30 min. FoxO3a, PP2A, and p27 expression levels were then measured. GAPDH was used as a loading control. Un, untreated cells; BA, β1-integrin blocking antibody pretreated cells. G, serum-starved human lung fibroblasts were preincubated with β1-integrin blocking antibody (1 μg/ml) in the presence of 10, 100, or 200 nm or absence of okadaic acid for 60 min. Cells were then attached to type I collagen for 30 min. Western analysis was carried out to measure p27 expression levels. GAPDH was used as a loading control. s/s, serum-starved human lung fibroblasts; DMSO, control.
    Figure Legend Snippet: FoxO3a increases p27 on the collagen matrix. A, serum-starved human lung fibroblasts were attached to collagen-coated plates as a function of time and p27 expression levels were measured. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. B, upper, FoxO3a siRNA (10 nm) transfected lung fibroblasts were serum-starved (s/s), and Western analysis was carried out to measure FoxO3a and p27 levels. GAPDH was used as a loading control. Lower, lung fibroblasts were ligated with β1-integrin activating antibody (TS2/16, 3 μg/ml) for 30 min in the presence or absence of FoxO3a siRNA. Western analysis was then carried out with antibodies as indicated. F3, FoxO3a siRNA; con, control siRNA; Un, untransfected cells. C, FoxO3a+/+ and Foxo3−/− cells were serum-starved for 1 day, and FoxO3a and p27 expression levels were measured using Western analysis. GAPDH was used as a loading control. D, FoxO3a+/+ cells were transfected with mouse FoxO3a siRNA (F3, 10 nm) or control siRNA (Con, 10 nm) and Western analysis was then carried out to measure FoxO3a and p27 expression levels. GAPDH was used as a loading control. Un, untransfected cells. E, serum-starved FoxO3a+/+ and FoxO3a−/− cells were attached to type I collagen for 30 min. p27 expression levels were then measured. GAPDH was used as a loading control. s/s, serum-starved fibroblasts. F, serum-starved human lung fibroblasts were preincubated with either β1-integrin blocking antibody (1 μg/ml) or isotype control mouse IgG (1 μg/ml) for 45 min. Cells were then attached to collagen-coated plates for 30 min. FoxO3a, PP2A, and p27 expression levels were then measured. GAPDH was used as a loading control. Un, untreated cells; BA, β1-integrin blocking antibody pretreated cells. G, serum-starved human lung fibroblasts were preincubated with β1-integrin blocking antibody (1 μg/ml) in the presence of 10, 100, or 200 nm or absence of okadaic acid for 60 min. Cells were then attached to type I collagen for 30 min. Western analysis was carried out to measure p27 expression levels. GAPDH was used as a loading control. s/s, serum-starved human lung fibroblasts; DMSO, control.

    Techniques Used: Expressing, Transfection, Western Blot, Blocking Assay

    FoxO3a suppresses cell proliferation. A, FoxO3a siRNA or control siRNA-transfected human lung fibroblasts were grown in 96-well plates and an MTS assay was carried out as described under “Materials and Methods.” Note that FoxO3a siRNA-transfected fibroblasts had an enhanced proliferation rate compared with that of control siRNA or untransfected cells. *, p < 0.03 versus control siRNA. B, PTEN+/+ cells infected with adenovirus expressing dominant-negative FoxO3a (DN) or empty vector (Vec) were grown in 96-well plates and an MTS assay was carried out (upper panel). Dominant-negative FoxO3a expression was evaluated using Western analysis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control (middle panel). PTEN+/+ cells infected with adenovirus expressing dominant-negative FoxO3a or empty vector were incubated with DMEM and cells numbers were measured at 24 h (lower panel). *, p < 0.05 versus empty vector. C, 3 × 104 of FoxO3a+/+ and FoxO3a−/− cells were serum-starved for 1 day followed by attachment to type I collagen-coated plates for 24 h. Cells numbers were then measured. D, FoxO3a−/− cells were infected with adenovirus expressing wild type FoxO3a (FoxO3a) or empty vector (GFP) along with uninfected (Un) cells for 24 h. 1.2 × 104 of Foxo3a−/− fibroblasts were then plated on tissue culture plates in the presence of 0.5% serum for 72 h. Cell numbers were then measured using a Coulter counter. Upper, Western analysis of FoxO3a protein expression in FoxO3a−/− cells infected with adenovirus expressing FoxO3a wild type or empty vector or untransfected cells. GAPDH was used as a loading control. Lower, % change in cell numbers in FoxO3a−/− cells infected with adenovirus expressing FoxO3a wild type or empty vector or untransfected cells. *, p < 0.03 versus GFP empty vector.
    Figure Legend Snippet: FoxO3a suppresses cell proliferation. A, FoxO3a siRNA or control siRNA-transfected human lung fibroblasts were grown in 96-well plates and an MTS assay was carried out as described under “Materials and Methods.” Note that FoxO3a siRNA-transfected fibroblasts had an enhanced proliferation rate compared with that of control siRNA or untransfected cells. *, p < 0.03 versus control siRNA. B, PTEN+/+ cells infected with adenovirus expressing dominant-negative FoxO3a (DN) or empty vector (Vec) were grown in 96-well plates and an MTS assay was carried out (upper panel). Dominant-negative FoxO3a expression was evaluated using Western analysis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control (middle panel). PTEN+/+ cells infected with adenovirus expressing dominant-negative FoxO3a or empty vector were incubated with DMEM and cells numbers were measured at 24 h (lower panel). *, p < 0.05 versus empty vector. C, 3 × 104 of FoxO3a+/+ and FoxO3a−/− cells were serum-starved for 1 day followed by attachment to type I collagen-coated plates for 24 h. Cells numbers were then measured. D, FoxO3a−/− cells were infected with adenovirus expressing wild type FoxO3a (FoxO3a) or empty vector (GFP) along with uninfected (Un) cells for 24 h. 1.2 × 104 of Foxo3a−/− fibroblasts were then plated on tissue culture plates in the presence of 0.5% serum for 72 h. Cell numbers were then measured using a Coulter counter. Upper, Western analysis of FoxO3a protein expression in FoxO3a−/− cells infected with adenovirus expressing FoxO3a wild type or empty vector or untransfected cells. GAPDH was used as a loading control. Lower, % change in cell numbers in FoxO3a−/− cells infected with adenovirus expressing FoxO3a wild type or empty vector or untransfected cells. *, p < 0.03 versus GFP empty vector.

    Techniques Used: Transfection, MTS Assay, Infection, Expressing, Dominant Negative Mutation, Plasmid Preparation, Western Blot, Incubation

    foxo3a  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc foxo3a
    Foxo3a, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/foxo3a/product/Cell Signaling Technology Inc
    Average 97 stars, based on 1 article reviews
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    foxo3a - by Bioz Stars, 2023-02
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