cdk2 m 2  (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: International journal of cancer. Journal international du cancer

Article Title: Mismatch repair system decreases cell survival by stabilizing the tetraploid G1 arrest in response to SN-38

doi: 10.1002/ijc.24893

Figure Lengend Snippet: The suppression of Cdk2 or of Cdk4 increases the tetraploid G1 arrest induced by SN-38 treatment. The suppression of Cdk2 or of Cdk4 in mock transfectants increases the tetraploid G1 arrest at day 6. Representative for 3 experiments. The suppression of Cdk6 had no detectable effect on the tetraploid arrest (not shown).

Article Snippet: For Cdk2 kinase activity, rabbit anti-Cdk2-agarose (M-2) (Santa Cruz) was used.

Techniques:

Journal: International journal of cancer. Journal international du cancer

Article Title: Mismatch repair system decreases cell survival by stabilizing the tetraploid G1 arrest in response to SN-38

doi: 10.1002/ijc.24893

Figure Lengend Snippet: The suppression of Cdk2 or of Cdk4 increases the tetraploid G1 arrest induced by SN-38 treatment. The suppression of Cdk2 or of Cdk4 in mock transfectants increases the tetraploid G1 arrest at day 6. Representative for 3 experiments. The suppression of Cdk6 had no detectable effect on the tetraploid arrest (not shown).

Article Snippet: For Cdk2 kinase activity, rabbit anti-Cdk2-agarose (M-2) (Santa Cruz) was used.

Techniques:

Journal: Cancer research

Article Title: MicroRNAs 221 and 222 Bypass Quiescence and Compromise Cell Survival

doi: 10.1158/0008-5472.CAN-07-6754

Figure Lengend Snippet: p57 3′UTR and p27 3′UTR are targets of miR-221 and miR-222. A, p57 and p27 mRNA 3′ UTRs depicting putative target sites for miR-221 and miR-222. The 3′ UTRs of p57 and p27 contain one and two sites expected to be recognized by both miR-221 and miR-222, respectively (arrowheads). Numbers represent the position of the seed match within the UTR sequences. Hs, human; Mm, mouse; Rn, rat; Cf, dog; Gg, chicken. B, repression of activity of the p57 3′ UTR-luciferase and p27 3′ UTR-luciferase constructs by ectopic expression of miR-221 and miR-222 oligonucleotides in K562 cells. Transfection of a nonfunctional miRNA (miR-NC2) was used as a negative control. Firefly luciferase activity was determined 48 h after transfection and normalized to Renilla luciferase activity from a promoter-less vector. Statistical significance was determined by the Student’s t test for three independent experiments (**, P < 0.01). C, K562 cells were either mock-transfected or transfected with miR-NC2, miR-221, miR-222, or a combination of miRNA and a 5-fold excess of the corresponding anti-miRNA inhibitor for 48 h. Total protein extracts were used to analyze p57 and p27 levels by Western blot with the specific antibodies (right). Transfection with miR-221 and miR-222 significantly reduced the levels of endogenous p57 and p27 proteins (lanes 3 and 7) when compared with mock (lanes 1 and 5) or miR-NC2 transfection (lanes 2 and 6). This decrease was reverted by the corresponding miRNA inhibitors (lanes 4 and 8). CDK2 is shown as loading control.

Article Snippet: The following dilutions of primary antibodies used were p57 C-20 rabbit polyclonal 1:1,000, cyclin A C-19 rabbit polyclonal 1:2,000, and CDK2 M-2 rabbit polyclonal 1:5,000 from Santa Cruz Biotechnology, Inc.; p27 mouse monoclonal IgG1 1:1,000 from BD Biosciences; and caspase-3 Asp175 rabbit polyclonal 1:1,000, caspase-8 1C12 mouse monoclonal 1:1,000, and caspase-9 rabbit polyclonal 1:1,000 from Cell Signaling.

Techniques: Activity Assay, Luciferase, Construct, Expressing, Transfection, Negative Control, Plasmid Preparation, Western Blot

Journal: Cancer research

Article Title: MicroRNAs 221 and 222 Bypass Quiescence and Compromise Cell Survival

doi: 10.1158/0008-5472.CAN-07-6754

Figure Lengend Snippet: Elevated levels of miR-221/miR-222 drive cells into S phase in the absence of serum growth factors. A, cell cycle distribution of T98G cells transfected with 75 nmol/L miRNAs for 24 h and then serum-deprived for 24 h. Cell cycle distribution was analyzed by FACS sorting, and profiles along with the percentage in each cell cycle stage are shown underneath. Cells transfected with miR-222 show accumulation of cells in S phase (asterisk), as well as a sub-2N peak (arrowhead). B, western blot analysis of samples taken from A confirmed the decrease in p27 protein level in miR-222–treated T98G cells; an increase in cyclin A protein is also observed, consistent with increased S-phase accumulation. Right, antibodies used. CDK2 was used as loading control. Asy, asynchronous.

Article Snippet: The following dilutions of primary antibodies used were p57 C-20 rabbit polyclonal 1:1,000, cyclin A C-19 rabbit polyclonal 1:2,000, and CDK2 M-2 rabbit polyclonal 1:5,000 from Santa Cruz Biotechnology, Inc.; p27 mouse monoclonal IgG1 1:1,000 from BD Biosciences; and caspase-3 Asp175 rabbit polyclonal 1:1,000, caspase-8 1C12 mouse monoclonal 1:1,000, and caspase-9 rabbit polyclonal 1:1,000 from Cell Signaling.

Techniques: Transfection, Western Blot

Journal: Cancer research

Article Title: MicroRNAs 221 and 222 Bypass Quiescence and Compromise Cell Survival

doi: 10.1158/0008-5472.CAN-07-6754

Figure Lengend Snippet: miR-221 and miR-222 induce apoptosis in the absence of growth factors. A, BrdUrd incorporation was monitored by immunofluorescence microscopy after transfection of T98G cells with miRNA oligonucleotides (75 nmol/L) for 24 h (left) and serum-deprived for 8 h. Note the presence of lobulated nuclei indicative of apoptosis. B, quantitative analysis of BrdUrd-positive cells in A (data not shown). Transfection of miR-221, miR-222, and the combination miR-221/miR-222 oligonucleotides shows ~76% of BrdUrd-positive cells compared with ~39% in miRNA negative control (miR-NC1) at both 8 and 24 h (>400 cells were counted). C, analysis of caspase activation. Samples treated as in A were resuspended in loading buffer and analyzed by Western blot for caspase cleavage products. Left, antibodies used. CDK2 was used as loading control.

Article Snippet: The following dilutions of primary antibodies used were p57 C-20 rabbit polyclonal 1:1,000, cyclin A C-19 rabbit polyclonal 1:2,000, and CDK2 M-2 rabbit polyclonal 1:5,000 from Santa Cruz Biotechnology, Inc.; p27 mouse monoclonal IgG1 1:1,000 from BD Biosciences; and caspase-3 Asp175 rabbit polyclonal 1:1,000, caspase-8 1C12 mouse monoclonal 1:1,000, and caspase-9 rabbit polyclonal 1:1,000 from Cell Signaling.

Techniques: Immunofluorescence, Microscopy, Transfection, Negative Control, Activation Assay, Western Blot

Journal: PLoS ONE

Article Title: Water Extract of Ashwagandha Leaves Has Anticancer Activity: Identification of an Active Component and Its Mechanism of Action

doi: 10.1371/journal.pone.0077189

Figure Lengend Snippet: ( A ) Western analysis of U2OS and TIG-1 cells for indicated proteins. Increase in p53 and p21 was observed both in cancer and normal cells. Whereas phosphorylated RB decreased in cancer cells, it increased in normal cells. Quantitation of the signals from three independent experiments is shown in the lower panel. ( B ) Changes in the level of cyclins (B1, D1 and E), CDK2, CDK4 and CDK6 are shown. Whereas cyclin D1 showed accumulation in cancer cells in response to TEG and WEX treatment; it was decreased in normal cells when treated in parallel. Quantitation of the signals from three independent experiments is shown in the lower panel. ( C ) Phosphorylation of RB as examined by immunostaining is shown. Whereas cancer cells showed decrease in phosphorylated RB, normal cells showed its increase. ( D ) WEX and TEG treated cancer cells, but not the normal cells, showed sharp decrease in MMP-9 and MMP-3. Quantitation of the signals from three independent experiments is shown in the lower panel.

Article Snippet: Immunoassays were done with anti-p53 (DO-1), -p21 (C-19), -CDK6 (C-21), -CDK4 (C-22), -CDK2 (M-2), -MMP9 (C-20), -MMP3 (C-19), -MMP2 (K-20), -cyclin B1 (H-433), -cyclin D1 (A-12), -cyclin E1 (M-20) antibodies (Santa Cruz), anti-γH2AX antibody (Millipore), anti-pRb antibody (ser-780) (Cell Signaling) and anti-actin antibody (Chemicon International, CA).

Techniques: Western Blot, Quantitation Assay, Phospho-proteomics, Immunostaining