PMC

FGF causes an immediate, transient delay in mitotic entry in RCS cells. (A) FACScan™ analysis of RCS cells treated with FGF1 and harvested at the indicated periods of time. Mitotic cells were stained with anti-phospho-histone H3(S10) antibodies. Numbers on the Y-axis indicate percentage of total cells in G₁, S, G₂ and M phases. (B) RCS cells were treated with either Nocodazole alone or together with FGF1. Only changes in the levels of G₂ and M phases are indicated. The data are representatives of several independent experiments with the same results.

FGF mediates CDC25C inactivation in RCS cells. (A) Western blot analysis of extracts (50 µg of total cellular protein) from RCS cells; untreated, Nocodazole-treated for 4 h or treated with lambda phosphatase after lysis. An upper band that is sensitive to lambda phosphatase treatment corresponds to a hyperphosphorylated form of CDC25C. (B–D) RCS cells were treated with FGF1 for the indicated times either in absence (B) or presence of Nocodazole (C and D). 50 µg of total cellular protein was analyzed by SDS-PAGE followed by immunoblotting with anti-CDC25C antibodies. Equal amount of protein loading was confirmed by α-actin immunodetection.

FGF-induced G₂ arrest is independent from the sequential sustained G₀/G₁ block. (A–D) Cell cycle arrest of RCS cells was induced by serum starvation for 32 h followed by release into medium containing 10% FBS and Nocodazole. (B–D) RCS cells were treated with FGF1 as indicated and subjected to cell cycle analysis. (E) Cyclin D1 and CDK4 were stably introduced into RCS cells, and this cell line and parental RCS cells were treated with FGF1 for the indicated periods of time. Kinase activity of immunoprecipitated cyclin B1/CDK1 complexes was assayed in vitro. The cyclin B1/CDK1 complexes were isolated from 1 mg of total cellular protein using anti-cyclin B1 antibodies. Histone H1 was used as a substrate.

FGF signaling strongly downregulates activity of cyclin B1/CDK1 complexes in chondrocytes. RCS cells were treated with FGF1 for indicated times. (A and C) Kinase activity of immunoprecipitated cyclin B1/CDK1 complexes was assayed in vitro. The cyclin B1/CDK1 complexes were isolated from 1 mg of total cellular protein using anti-cyclin B1 antibodies, and histone H1 was used as a substrate. Antimouse IgG was used as a negative control. An equal amount of protein loading was confirmed by immunodetection of cyclin B1 and CDK1 in immunoprecipitated complexes. (B) 20 µg of total cellular protein were analyzed by SDS-PAGE followed by immunoblotting for anti-phospho-CDK1(Y15), anti-CDK1 and anti-phospho-CDK1(T14) antibodies. Equal amount of protein loading was confirmed by α-tubulin and α-actin immunodetection. (C) RCS cells were pretreated with Nocodazole for 12 hrs and then FGF1 was added for indicated periods of time.

FGF signaling modulates the activity of Myt1 kinase. RCS cells were treated with FGF1 for indicated periods of times. (A) 20 µg of total cellular protein were analyzed by SDS-PAGE followed by immunoblotting for anti-Wee1 and anti-Myt1 antibodies. Equal amount of protein loading was confirmed by α-tubulin immunodetection. (B and C) Lysates were prepared and normalized by the amount of total cellular protein and subjected to immunoprecipitation with anti-Myt1 antibody as described in Materials and Methods. 10% of input and 50% of immunoprecipitated samples were analyzed by immunoblotting with anti-Myt1, anti-CDK1 or anti-phospho-CDK1(Y15) antibody. Control immunoprecipitation (*) was done using agarose A beads. (C) Kinase activity of immunoprecipitated Myt1 complexes was assayed in vitro. Recombinant cyclin B1/CDK1 complex was used as a substrate for Myt1 immunoprecipitates and phosphorylation of CDK1 was monitored by immunoblotting using anti-phospho-CDK1(Y15) antibody. Note that under these conditions endogenous CDK1 and phospho-CDK1(Y15) were not detectable in Myt1 immunoprecipitates.

FGF-induced inhibition of CDC25C phosphorylation is not mediated by activation of a phosphatase. (A) RCS cells were pre-incubated with okadaic acid (OA) at the indicated concentrations for 1 hour and either treated or not with FGF1 for one more hour. 50 µg of total cellular protein were analyzed by SDS-PAGE, followed by immunoblotting for anti-CDC25C antibodies. Equal amount of protein loading was confirmed by α-actin immunodetection. (B) RCS cells were infected with adenoviruses expressing either GFP or SV40 ST antigen following FGF1 treatment as indicated. Kinase activity of immunoprecipitated cyclin B1/CDK1 complexes was assayed in vitro. The cyclin B1/CDK1 complexes were isolated from 0.5 mg of total cellular protein using anti-cyclin B1 antibodies. Histone H1 was used as a substrate. Antimouse IgG was used as a negative control. RCS cells infected with SV40 ST routinely exhibited lower basal levels of cyclin B1-associated kinase activity in untreated cells as assayed in several independent experiments. (C) RCS cells were treated with Nocodazole and either with CDK1 inhibitor (Alsterpaullone), MEK1/2 inhibitor (U0126) or PLK1/3 inhibitor (GW843682X), as marked, and harvested at the indicated times. 50 µg of total cellular protein were analyzed by SDS-PAGE followed by immunoblotting for anti-CDC25C antibodies.