α-cdk2 (m-2) antibody  (Santa Cruz Biotechnology)

Journal: BMC Molecular and Cell Biology

Article Title: Human embryonic stem cells display a pronounced sensitivity to the cyclin dependent kinase inhibitor Roscovitine

doi: 10.1186/s12860-019-0222-3

Figure Lengend Snippet: ROSC affects cell cycle distribution and key cell cycle regulators expression. a hESCs were exposed to ROSC or DMSO for 16 h. Cell cycle distribution of asynchronously growing or ROSC-treated H9 and HF was determined by flow cytometry analysis of cellular DNA content following cell staining with PI (left panel). Cells were pulse labeled with BrdU for 30 min prior to harvesting and stained with anti-BrdU-APC conjugate and with7-amino-actinomycin D (7-AAD) for determination of DNA synthesis and DNA content respectively (right panel). Bar graphs summarizing flow cytometry cell cycle profile analysis. Error bars represent means ± SD from three independent experiments (lower panels). b Comparison of mRNA expression levels for cyclin D1, D2, E, A2, B1 and B2 in hESCs and HF assessed by Real Time RT-PCR (left panel). Rpl7 expression served as normalizer. Graph shows mRNA fold change relative to HF. The mean ± SEM from three independent experiments are shown. c Comparison of mRNA expression levels of cdk1, cdk2, cdk4 and cdk6 in hESCs and HF analyzed by Real Time RT-PCR (left panel). Representative Western blot images of CDK2, CDK4 and CDK6 (right panel). β-Tubulin served as loading control. Bar graphs show densitometric quantification. Data are expressed as means ± SD (left panel). d Time course analysis of mRNA levels of cyclin D1, D2, E, A2, B1, B2 and cdk1, cdk2, cdk7 and cdk9 were assessed by Real Time RT-PCR in ROSC-treated or untreated hESCs. Rpl7 expression served as normalizer. Graph shows mRNA fold change relative to untreated cells. The mean ± SEM from three independent experiments are shown. In all cases paired Student’s t test was used to test for significant differences * P < 0.05, *** P < 0.001

Article Snippet: The following primary antibodies were used: α-Actin (sc-1616), α-Bax (N-20) (sc-493), α-BclX S / L (S-18) (sc-634), α-Mcl-1(S-19) (sc-819), α-CDK2 (M-2) (sc-163), α-CDK4 (H22) (sc-601), α-CDK6 (C-21) (sc-177) (Santa Cruz Biotechnology), α-phospho-p53serine46 (cat.2521) (Cell Signaling Technology, Beverly, MA, USA), α-p53 (DO-1) (ab1101) (Abcam Inc., Cambridge, MA, USA).

Techniques: Expressing, Flow Cytometry, Staining, Labeling, DNA Synthesis, Comparison, Quantitative RT-PCR, Western Blot, Control

Journal: BMC Molecular and Cell Biology

Article Title: Human embryonic stem cells display a pronounced sensitivity to the cyclin dependent kinase inhibitor Roscovitine

doi: 10.1186/s12860-019-0222-3

Figure Lengend Snippet: Small molecule inhibition of CDK2 did not induce cell death in hESCs. a Representative histograms of PI stained cells exposed or not to CDK2 inhibitor II (5 μM) for 24 h. Percentage of PI+ cells was determined by flow cytometric analysis (top panel). Representative photomicrographs of H9 colonies and HF treated or not with 5 μM CDK2 inhibitor II over a 24 h period (bottom left panel). p53 and Mcl-1protein levels were analyzed in the presence or absence of CDK2 inhibitor by Western blot. GAPDH and Actin were used as loading controls. Bar graphs show densitometric quantification (bottom right panel). b Pluripotency markers oct-4 and nanog mRNA expression levels by Real Time RT-PCR in CDK2 inhibitor II-treated or untreated (vehicle) hESCs. Rpl7 expression was used as normalizer (right panel). The mean ± SEM from three independent experiments are shown. In all cases, a paired Student’s t test was used to test for significant differences between ROSC-treated and untreated samples. * P < 0.05. Immunofluorescence photomicrographs of CDK2 inhibitor II-treated H9 cells (5 μM during 72 h). Representative images of H9 cells stained with primary antibody against Oct-4. Nuclei were counterstained with DAPI. Scale bars represent 100 μm

Article Snippet: The following primary antibodies were used: α-Actin (sc-1616), α-Bax (N-20) (sc-493), α-BclX S / L (S-18) (sc-634), α-Mcl-1(S-19) (sc-819), α-CDK2 (M-2) (sc-163), α-CDK4 (H22) (sc-601), α-CDK6 (C-21) (sc-177) (Santa Cruz Biotechnology), α-phospho-p53serine46 (cat.2521) (Cell Signaling Technology, Beverly, MA, USA), α-p53 (DO-1) (ab1101) (Abcam Inc., Cambridge, MA, USA).

Techniques: Inhibition, Staining, Western Blot, Expressing, Quantitative RT-PCR, Immunofluorescence

Journal:

Article Title: Cell Cycle Arrest and Cell Death Are Controlled by p53-dependent and p53-independent Mechanisms in Tsg101-deficient Cells *

doi: 10.1074/jbc.M400408200

Figure Lengend Snippet: A, flow cytometric analysis of the DNA content of viable Tsg101-deficient MEFs (Tsg101fl/fl, pBabe-Cre) that lack one or two alleles of the p53 gene (p53+/− and p53−/ −) and their uninfected controls (Tsg101fl/fl p53−/ − ). The sub-G1 population of apoptotic cells was gated out in this assay. Note that the relative number of cells in S phase is reduced only in p53 heterozygous knock-out cells lacking Tsg101 but not in Tsg101-deficient MEFs carrying two mutant p53 alleles. B, Western blot analysis of cyclin A (S-phase cyclin) and regulators of G1/S progression (p19Arf and p21Cip) in Tsg101-deficient MEFs that lack one or two copies of p53 and their uninfected controls. Note that cyclin A2 was markedly down-regulated in Tsg101-deficient MEFs that carry at least one functional allele of p53. Immortal p53/p21Cip1-deficient MEFs express high levels of cyclin A2 and p19Arf regardless of the Tsg101 mutation status. C, Cdk2 activity assay using histone H1 as a substrate for phosphorylation. Only Tsg101 knock-out MEFs that carry at least one functional p53 allele exhibit a reduced activity of Cdk2. The complete deletion of p53 and, subsequently, the absence of the Cdk2 inhibitor p21Cip1 restore a normal activity of Cdk2 in Tsg101-deficient cells.

Article Snippet: A Cdk2 kinase assay was performed using the α-Cdk2 (M-2) antibody from Santa Cruz Biotechnology and Histone H1 (1 μg/μl) from Sigma.

Techniques: Knock-Out, Mutagenesis, Western Blot, Functional Assay, Activity Assay