Journal: PLoS ONE

Article Title: Brain-Derived Neurotrophic Factor-Dependent cdk1 Inhibition Prevents G2/M Progression in Differentiating Tetraploid Neurons

doi: 10.1371/journal.pone.0064890

Figure Lengend Snippet: ( A ) Lysates from DCRNs cultured for 20 h in the presence of different combinations of 100 ng/m NGF and 2 ng/ml BDNF were subjected to western blot with antibodies specific for either cdk1 (upper panel) or β-actin (lower panel). ( B ) Normalized cdk1/β-actin ratio. ( C ) Semiquantitative RT-PCR analysis of mRNA samples obtained from DCRNs cultured for 20 h in the presence of either vehicle (Control) or 2 ng/ml BDNF (BDNF). The levels of Cdk1 expression were normalized to Gapdh . N.S. non-significant; *p<0.05 (Student’s t test; n = 3–4).

Article Snippet: Cultures were maintained at 37°C for 20 h in a water-saturated atmosphere containing 5% CO 2 and where stated, they were supplemented with recombinant NGF (Alomone Labs, and Sigma) and/or BDNF (Alomone Labs) for different time periods.

Techniques: Cell Culture, Western Blot, Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: PLoS ONE

Article Title: Brain-Derived Neurotrophic Factor-Dependent cdk1 Inhibition Prevents G2/M Progression in Differentiating Tetraploid Neurons

doi: 10.1371/journal.pone.0064890

Figure Lengend Snippet: ( A ) General cdk protein kinase activity, based on the phosphorylation of a Rb-derived peptide by the cdk activity present in cell extracts from DCRNs cultured for 20 h in the presence of 100 ng/ml NGF and treated for 30 min with either vehicle (Control) or 2 ng/ml BDNF. Equal amount of cell extracts were used in both cases. ( B ) Left panel, a representative western blot performed with an anti-cdk1 antibody using the cell extracts described above prior to immunoprecipitation (INPUT). Right panel, protein kinase activity immunoprecipitated with an anti cdk1 antibody (i.e. cdk1-specific kinase activity) from the cell extracts described above, normalized to the relative amount of cdk1 present in these extracts (see input in left panel). *p<0.05 (ANOVA; n = 3).

Article Snippet: Cultures were maintained at 37°C for 20 h in a water-saturated atmosphere containing 5% CO 2 and where stated, they were supplemented with recombinant NGF (Alomone Labs, and Sigma) and/or BDNF (Alomone Labs) for different time periods.

Techniques: Activity Assay, Derivative Assay, Cell Culture, Western Blot, Immunoprecipitation

Journal: PLoS ONE

Article Title: Brain-Derived Neurotrophic Factor-Dependent cdk1 Inhibition Prevents G2/M Progression in Differentiating Tetraploid Neurons

doi: 10.1371/journal.pone.0064890

Figure Lengend Snippet: ( A ) E6 retinal cells electroporated with either EGFP (-) or cdk1 plus cyclin B1 (CC) and EGFP (+) were cultured under neurogenic conditions for 20 h in the presence of different combinations of 100 ng/ml NGF and 2 ng/ml BDNF. The percentage of mitotic figures was evaluated in the EGFP-positive cells. Lower panels show an example of a mitotic figure (pH3; red) in an EGFP-transfected cell (EGFP) (arrow). ( B ) E6 retinal cells were electroporated and cultured as above. The percentage of pyknotic nuclei was evaluated in the EGFP-positive cells. Lower panels show an example of a pyknotic nucleus in an EGFP-transfected cell (EGFP) (arrow). Bisb.: bisbenzimide. *p<0.05; ***p<0.005 (Student’s t test; n = 4). Bars: 10 µm.

Article Snippet: Cultures were maintained at 37°C for 20 h in a water-saturated atmosphere containing 5% CO 2 and where stated, they were supplemented with recombinant NGF (Alomone Labs, and Sigma) and/or BDNF (Alomone Labs) for different time periods.

Techniques: Cell Culture, Transfection

Journal: PLoS ONE

Article Title: Brain-Derived Neurotrophic Factor-Dependent cdk1 Inhibition Prevents G2/M Progression in Differentiating Tetraploid Neurons

doi: 10.1371/journal.pone.0064890

Figure Lengend Snippet: ( A ) Validation of the cdk1 sandwich ELISA assay performed with 10 fg of either GST (GST) or a chimeric protein containing the sequence of human cdk1 bound to GST (Cdk1-GST), and revealed with the anti-cdk1 PSTAIRE antibody. Normalized optical density (O.D.) at 405 nm is shown. ( B ) Validation of the phospho-cdk1 sandwich ELISA assay performed with cell lysates obtained from DCRNs cultured in the presence of 100 ng/ml NGF and 2 ng/ml BDNF, incubated either in the absence (- CIP) or presence (+ CIP) of CIP, and revealed with the anti-cdk1 (pTyr15) antibody. Normalized optical density (O.D.) at 405 nm is shown. ( C ) Cell lysates from DCRNs cultured in the presence (+) or absence (-) of the referred factors were subjected to sandwich ELISA analyses using antibodies specific for either cdk1 phosphorylated at Tyr15 or total cdk1 protein. Colorimetric values for cdk1 phosphorylated at Tyr15 were normalized to the levels of cdk1 protein (Cdk1). ( D ) Cell lysates from DCRNs cultured in the presence of the referred factors and/or K252a were subjected to sandwich ELISA analyses as described above. Colorimetric values for cdk1 phosphorylated at Tyr15 were normalized to the levels of cdk1 protein (Cdk1). *p<0.05; ***p<0.005 (Student’s t test; n = 3).

Article Snippet: Cultures were maintained at 37°C for 20 h in a water-saturated atmosphere containing 5% CO 2 and where stated, they were supplemented with recombinant NGF (Alomone Labs, and Sigma) and/or BDNF (Alomone Labs) for different time periods.

Techniques: Sandwich ELISA, Sequencing, Cell Culture, Incubation

Journal: PLoS ONE

Article Title: Brain-Derived Neurotrophic Factor-Dependent cdk1 Inhibition Prevents G2/M Progression in Differentiating Tetraploid Neurons

doi: 10.1371/journal.pone.0064890

Figure Lengend Snippet: ( A ) Cell lysates from DCRNs cultured in the presence of the referred factors and/or 300 nM MK-1775 were subjected to sandwich ELISA analyses using antibodies specific for either cdk1 phosphorylated at Tyr15 or total cdk1 protein. Colorimetric values for cdk1 phosphorylated at Tyr15 were normalized to the levels of cdk1 protein (Cdk1). ( B ) Cell lysates from CEFs cultured in the presence (+) or absence (-) of 300 nM MK-1775 were subjected to sandwich ELISA analyses as described above. Colorimetric values for cdk1 phosphorylated at Tyr15 were normalized to the levels of cdk1 protein (Cdk1). ***p<0.005 (Student’s t test; n = 3). # p<0.05 (NGF/BDNF/MK-1775 vs NGF/MK-1775; Student’s t test; n = 3).

Article Snippet: Cultures were maintained at 37°C for 20 h in a water-saturated atmosphere containing 5% CO 2 and where stated, they were supplemented with recombinant NGF (Alomone Labs, and Sigma) and/or BDNF (Alomone Labs) for different time periods.

Techniques: Cell Culture, Sandwich ELISA

Journal: PLoS ONE

Article Title: Brain-Derived Neurotrophic Factor-Dependent cdk1 Inhibition Prevents G2/M Progression in Differentiating Tetraploid Neurons

doi: 10.1371/journal.pone.0064890

Figure Lengend Snippet: E6 retinal cells electroporated with either EGFP (-) or the Tyr15Phe mutant form of cdk1 plus cyclin B1 (Mut CC) and EGFP (+) were cultured for 20 h under neurogenic conditions in the presence of different combinations of 100 ng/ml NGF and 2 ng/m BDNF. The percentage of pyknotic nuclei was evaluated in EGFP-positive cells. *p<0.05 (Student’s t test; n = 4).

Article Snippet: Cultures were maintained at 37°C for 20 h in a water-saturated atmosphere containing 5% CO 2 and where stated, they were supplemented with recombinant NGF (Alomone Labs, and Sigma) and/or BDNF (Alomone Labs) for different time periods.

Techniques: Mutagenesis, Cell Culture