Journal: The Journal of Cell Biology

Article Title: Dual Stimulation of Ras/Mitogen-Activated Protein Kinase and Rhoa by Cell Adhesion to Fibronectin Supports Growth Factor–Stimulated Cell Cycle Progression

doi:

Figure Lengend Snippet: Fibronectin specifically supports E2F-controlled S phase entry. (A) Serum-starved cells were plated on agar in suspension (sus), poly- l -lysine (PLL), laminin 1 (LN), collagen 1 (COL), or fibronectin (FN). 16 h after growth factor addition, cells were lysed and monitored for pRb phosphorylation by immunoprecipitation followed by Western blotting with Rb antibodies. (B) 20 h after growth factor addition, luciferase activity was measured in cells stably expressing a wild-type cyclin A luciferase reporter (black bars) or a cyclin A reporter harboring a mutated E2F site (white bars). (C) Cells stably expressing a wild-type cyclin A luciferase reporter were transfected with a puromycin resistance vector combined with an empty pcDNA vector (mock) or with pcDNA containing a nonphosphorylatable Rb mutant (Rb(NPC−)). After selection for transfectants in medium containing puromycin, the cells were plated on fibronectin. 20 h after growth factor addition, luciferase activity was measured.

Article Snippet: Luciferase reporter constructs for cyclin A (cyclin A gene promoter region from −7300 to +11 relative to the major transcription start site) , cyclin A with a mutated E2F-binding site (harboring a mutated E2F site at −37 to −33) , cyclin D1 (promoter region from −973 to +139), and for p21 Cip/Waf (2.4-kb p21 promoter region) were gifts from Berthold Henglein (Institut Curie, Paris, France).

Techniques: Suspension, Phospho-proteomics, Immunoprecipitation, Western Blot, Luciferase, Activity Assay, Stable Transfection, Expressing, Transfection, Plasmid Preparation, Mutagenesis, Selection

Journal: The Journal of Cell Biology

Article Title: Dual Stimulation of Ras/Mitogen-Activated Protein Kinase and Rhoa by Cell Adhesion to Fibronectin Supports Growth Factor–Stimulated Cell Cycle Progression

doi:

Figure Lengend Snippet: Fibronectin stimulates RhoA activity in serum-starved cells. (A) Serum-starved cells were replated overnight on various substrates as indicated and analyzed for paxillin and actin localization by immunofluorescence. (B) Serum-starved cells were plated on the indicated substrates and lysed after 2 h for biochemical RhoA and Rac1 activity assays. Levels of bound RhoA or Rac1 (top) or total levels in a 5% aliquot of each lysate (bottom) were determined by Western blotting using RhoA- or Rac1-specific antibodies. (C) Serum-starved cells were plated overnight on the indicated substrates followed by incubation for 10 min in the presence of growth factors, then lysed for biochemical RhoA and Rac1 activity assays. In B and C, the percentage of GTP-bound RhoA and Rac1 was determined according to densitometry of autoradiograms. (D) Cells stably expressing a wild-type cyclin A luciferase reporter were transfected with a puromycin resistance vector combined with an empty vector (mock) or with a C3 expression plasmid and selected in medium containing puromycin. Subsequently, the cells were plated in suspension, on polylysine, or on fibronectin, and 20 h after growth factor addition, luciferase activity was measured. (E) Cells stably expressing a wild-type cyclin A luciferase reporter were transduced with dominant inhibitory RhoA or Rac1 or control retrovirus and selected in media containing zeocin. Subsequently, the cells were plated on fibronectin, and 20 h after growth factor addition luciferase activity was measured. The level of activity in mock-transduced cells was set at 1. The inset shows Western blot analysis of the Myc-tagged constructs.

Article Snippet: Luciferase reporter constructs for cyclin A (cyclin A gene promoter region from −7300 to +11 relative to the major transcription start site) , cyclin A with a mutated E2F-binding site (harboring a mutated E2F site at −37 to −33) , cyclin D1 (promoter region from −973 to +139), and for p21 Cip/Waf (2.4-kb p21 promoter region) were gifts from Berthold Henglein (Institut Curie, Paris, France).

Techniques: Activity Assay, Immunofluorescence, Western Blot, Incubation, Stable Transfection, Expressing, Luciferase, Transfection, Plasmid Preparation, Suspension, Transduction, Control, Construct

Journal: The Journal of Cell Biology

Article Title: Dual Stimulation of Ras/Mitogen-Activated Protein Kinase and Rhoa by Cell Adhesion to Fibronectin Supports Growth Factor–Stimulated Cell Cycle Progression

doi:

Figure Lengend Snippet: Regulation of ERK-type MAPKs. (A) Cells stably expressing a luciferase reporter for cyclin A or for cyclin D1 were transfected with a puromycin resistance vector combined with an empty vector (mock) or with a dominant inhibitory MEK expression plasmid and selected in medium containing puromycin. Subsequently, the cells were plated on fibronectin and lysed for luciferase activity assays 4 h (in the case of the cyclin D1 reporter) or 20 h (cyclin A reporter) after growth factor addition. (B) Serum-starved cells were plated overnight in suspension or on polylysine or fibronectin. They were then stimulated with growth factors and lysed at the indicated time points for Western blotting with antibodies against total ERK1 and ERK2 or with a phospho-ERK antibody. (C) Serum-starved cells were plated overnight on the indicated substrates and fixed for analysis of ERK2 localization by immunofluorescence before (−) or 2.5 h after growth factor stimulation (+).

Article Snippet: Luciferase reporter constructs for cyclin A (cyclin A gene promoter region from −7300 to +11 relative to the major transcription start site) , cyclin A with a mutated E2F-binding site (harboring a mutated E2F site at −37 to −33) , cyclin D1 (promoter region from −973 to +139), and for p21 Cip/Waf (2.4-kb p21 promoter region) were gifts from Berthold Henglein (Institut Curie, Paris, France).

Techniques: Stable Transfection, Expressing, Luciferase, Transfection, Plasmid Preparation, Activity Assay, Suspension, Western Blot, Immunofluorescence

Journal: The Journal of Cell Biology

Article Title: Dual Stimulation of Ras/Mitogen-Activated Protein Kinase and Rhoa by Cell Adhesion to Fibronectin Supports Growth Factor–Stimulated Cell Cycle Progression

doi:

Figure Lengend Snippet: Regulation of cyclin D1 and p21 Cip/Waf RNA. (A) Cells stably expressing a cyclin D1 (left) or a p21 Cip/Waf (right) luciferase reporter were plated in suspension (sus, filled circles), on poly-lysine (PLL, open circles), or on fibronectin (FN, filled squares) (time a) and stimulated with growth factors 16 h later (time b). At the indicated time points, luciferase activity was measured. (B) Cells were starved, plated overnight in suspension or on polylysine or fibronectin, and subsequently stimulated with growth factors. At the indicated time points, cells were lysed for Northern blotting with probes against mouse cyclin D1 or p21 Cip/Waf .

Article Snippet: Luciferase reporter constructs for cyclin A (cyclin A gene promoter region from −7300 to +11 relative to the major transcription start site) , cyclin A with a mutated E2F-binding site (harboring a mutated E2F site at −37 to −33) , cyclin D1 (promoter region from −973 to +139), and for p21 Cip/Waf (2.4-kb p21 promoter region) were gifts from Berthold Henglein (Institut Curie, Paris, France).

Techniques: Stable Transfection, Expressing, Luciferase, Suspension, Activity Assay, Northern Blot

Journal: The Journal of Cell Biology

Article Title: Dual Stimulation of Ras/Mitogen-Activated Protein Kinase and Rhoa by Cell Adhesion to Fibronectin Supports Growth Factor–Stimulated Cell Cycle Progression

doi:

Figure Lengend Snippet: Regulation of cyclin D1 and p21 Cip/Waf protein. (A) Morphology of pCEF/C3-transfected cells. (B) Cells were transfected with a puromycin resistance vector combined with empty vector (mock) or with a C3 toxin expression plasmid and selected for transfectants in medium containing puromycin. Subsequently, the cells were serum-starved, plated on polylysine or fibronectin, and stimulated with growth factors 16 h later and lysed at the indicated time points for Western blotting with antibodies against MAPK, phospho-MAPK, or cyclin D1. (C) Untransfected cells (left) or mock or pCEF/C3 transfected cells (right) were serum starved and plated on the indicated substrates. 16 h later they were stimulated with growth factors and lysed at the indicated time points for Western blotting with antibodies against p21 Cip/Waf .

Article Snippet: Luciferase reporter constructs for cyclin A (cyclin A gene promoter region from −7300 to +11 relative to the major transcription start site) , cyclin A with a mutated E2F-binding site (harboring a mutated E2F site at −37 to −33) , cyclin D1 (promoter region from −973 to +139), and for p21 Cip/Waf (2.4-kb p21 promoter region) were gifts from Berthold Henglein (Institut Curie, Paris, France).

Techniques: Transfection, Plasmid Preparation, Expressing, Western Blot

Journal: The Journal of Cell Biology

Article Title: Dual Stimulation of Ras/Mitogen-Activated Protein Kinase and Rhoa by Cell Adhesion to Fibronectin Supports Growth Factor–Stimulated Cell Cycle Progression

doi:

Figure Lengend Snippet: Model depicting proposed roles of growth factors and adhesion to fibronectin in regulation of G1 cell cycle progression. Cell adhesion and spreading on various substrates collaborates with soluble mitogens to stimulate activity of Rac and MAPK, which may be connected at the level of Ras. Growth factors and cell spreading can both induce transcription of cyclin D1 and p21 Cip/Waf . Cell adhesion to fibronectin, in addition to these pathways, also activates RhoA, which is required for the suppression of p21 Cip/Waf and the accumulation of cyclin D1 later in G1. We show that adhesion to fibronectin suppresses p21 Cip/Waf RNA levels whereas the stimulation of cyclin D1 induction occurs at the protein level. Together, these signaling pathways stimulate the hyperphosphorylation of Rb pocket proteins, cyclin A induction, and DNA synthesis.

Article Snippet: Luciferase reporter constructs for cyclin A (cyclin A gene promoter region from −7300 to +11 relative to the major transcription start site) , cyclin A with a mutated E2F-binding site (harboring a mutated E2F site at −37 to −33) , cyclin D1 (promoter region from −973 to +139), and for p21 Cip/Waf (2.4-kb p21 promoter region) were gifts from Berthold Henglein (Institut Curie, Paris, France).

Techniques: Activity Assay, Protein-Protein interactions, DNA Synthesis