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

Journal:

Article Title: Zbtb4 represses transcription of P21CIP1 and controls the cellular response to p53 activation

doi: 10.1038/emboj.2008.85

Figure Lengend Snippet: Depletion of Zbtb4 causes resistance to vincristine treatment through upregulation of P21CIP1. (A) Vincristine induces activation of p53. The panels show immunoblots of SH-EP cells infected with either a control vector (left) or a vector expressing a dominant-negative allele of p53 (p53DD; right) probed with the indicated antibodies. (B) Transient depletion of Zbtb4 upregulates P21CIP1 mRNA levels. The panels document the RQ–PCR analysis of relative mRNA expression level of ZBTB4, P21CIP1, 14-3-3σ, BTG2, GADD45, PUMA, BAX and NOXA in SH-EP and SH-SY5Y neuroblastoma cells transfected with either control or siRNA targeting ZBTB4. Cells were harvested 48 h after transfection and expression levels were determined by RQ–PCR. (C) Stable depletion of Zbtb4 upregulates p21Cip1 protein. The panels show immunoblots of p21Cip1 and Cdk2 protein levels in indicated SH-EP cell clones stably infected with either control vectors or vectors expressing shZBTB4. (D) Ectopic expression of Zbtb4 represses P21CIP1. The panels document the RQ–PCR analysis of the relative mRNA expression level of ZBTB4, P21CIP1, PUMA and NOXA in SH-EP cells stably infected with HA-tagged ZBTB4 vector or a control vector. (E) P21CIP1 is required for protection of SH-EP cells from vincristine-induced apoptosis by depletion of Zbtb4. The indicated SH-EP cell clones stably expressing either shZBTB4 or control shRNA were transiently transfected with vectors expressing shP21CIP1 or a scrambled shRNA. At 48 h after transfection, cells were plated in triplicate for vincristine treatment. Cells were treated with either vincristine or DMSO for 3 days; subsequently, the relative cell number was determined. Error bars represent standard error of the mean.

Article Snippet: Antibodies The following antibodies were used: α-HA (HA.11; Covance), α-p21Cip1 (N-20), α-p53 (DO-1), α-Cdk2 (M2), α-mSin3b (H4) (all Santa Cruz Biotechnology), α-β-tubulin (MAB3408; Chemicon), α-Miz1 (10E2, Herold et al , 2002 ; H190, Santa Cruz Biotechnology) and α-Zbtb4 ( Filion et al , 2006 ).

Techniques: Activation Assay, Western Blot, Infection, Control, Plasmid Preparation, Expressing, Dominant Negative Mutation, Transfection, Clone Assay, Stable Transfection, shRNA

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

Journal:

Article Title: ACTR/AIB1 Functions as an E2F1 Coactivator To Promote Breast Cancer Cell Proliferation and Antiestrogen Resistance

doi: 10.1128/MCB.24.12.5157-5171.2004

Figure Lengend Snippet: Stimulation of hormone-independent cell proliferation by ACTR requires ACTR interaction with E2F1, but not ER. (A and B) T-47D cells were infected with adenovirus expressing wild-type and mutant forms of ACTR, as depicted. The expression levels of different ACTR proteins in the cells 2 days after infection were determined by Western blotting using α-HA antibody. Cell proliferation in the absence of hormone was monitored by MTT assay 4 days after infection as described in the legend for Fig. ​Fig.2B,2B, and the percentage of wild-type ACTR activity was determined by dividing the proliferation induced by the mutant ACTR with that of wild-type ACTR. (C and D) ER-negative cells (HCC-1806 and HCC-1937) infected with adeno-ACTR or adeno-GFP were monitored for cell proliferation by MTT assay. (E) T-47D and its derivative, A41 cells, hormone deprived for 2 days, were infected with an equal titer of the RNAi adenoviruses for E2F1 or GFP, as for Fig. ​Fig.1A.1A. Cells were then replenished with hormone-depleted medium either with (T-47D) or without (A41) 10−7 M E2. Cell proliferation was determined at 6 days after infection, with the values from Ri-GFP adenovirus-infected cells set as 100 arbitrarily. Cells were also collected at day 6 postinfection for Western analysis using α-E2F1 and α-β-actin. Note that no significant effect of GFP-RNAi vector on cell proliferation was observed when compared to mock infection. (F) Potential pathways for elevated levels of ACTR to stimulate breast cancer cell proliferation.

Article Snippet: Western blotting analysis of proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed using the following specific antibodies: α-ACTR, α-TIF2, α-cyclin A, α-cyclin B, and α-Cdk4 (Transduction Labs); α-Cdk2 (M2), α-Cdc2 ( 17 ) α-cyclin D1 (H295), α-cyclin D2 (C17), α-cyclin E (HE12), α-SRC-1 (M341), α-p107 (C18), α-p130 (C20), α-p21 (M19), α-p27 (M197), α-E2F1 (KH95), α-E2F3 (C18), and α-E2F4 (C20; Santa Cruz); α-E2F2 (BD), α-Rb (IF8), and α-ERα (Ab-15; Neomarkers); and α-β-actin (Sigma).

Techniques: Infection, Expressing, Mutagenesis, Western Blot, MTT Assay, Activity Assay, Plasmid Preparation