Journal: Journal of bioinformatics and systems biology : Open access

Article Title: CDKs Functional Analysis in Low Proliferating Early-Stage Pancreatic Ductal Adenocarcinoma.

doi: 10.26502/jbsb.5107060

Figure Lengend Snippet: Figure 2: A. Log2 mRNA expression level of MKI67 in the high group (≥ 75th percentile) and low group (<75th percentile) in early-stage PDAC patients. B. Patients in the high MKI67 group (MKI67 high) had shorter median survival compared to the low MKI67 group (MKI67 low). Pearson's correlation analysis was performed to analyze the mRNA expression correlation between MKI67 and CDK1 (C), CDK2 (D), CDK4 (E), and CDK6 (F).

Article Snippet: PANC1 and MiaPaca II cells were treated with CDK1 inhibitors (10 uM CDK1-IN-2, MedChemExpress HY-112463; or 5 uM RO-3306, MedChemExpress HY12529) or vehicle for 24h or 48hrs in the presence/absence of 10%FBS.

Techniques: Expressing

Journal: Archives of oral biology

Article Title: A novel T-cell proliferation-associated gene predicts prognosis and reveals immune infiltration in patients with oral squamous cell carcinoma.

doi: 10.1016/j.archoralbio.2023.105719

Figure Lengend Snippet: Fig. 3. Development of a T-lymphocyte proliferation regulator-related prognostic signature. (a) Univariable Cox hazard analyses were completed on T-lymphocyte proliferation regulator pairs. (b) LASSO variable trajectory plots were screened for non-zero variables satisfying the lambda.min coefficient by ten-fold cross-vali dation. (c) Venn diagrams showed that T-lymphocyte proliferation regulators with statistical significance in univariable Cox hazard analyses and non-zero variables with lambda.min coefficients in LASSO variable trajectory plots and baseMean > 1000 that satisfied the above three conditions were screened out as the T- lymphocyte proliferation regulator signature (RAN, CDK1, CDK2) for prognostic risk model construction. (d) Curves of the LASSO coefficients for the three-T- lymphocyte proliferation regulator signature. (e, f) Differences in survival between high-risk and low-risk patients in the training and validation groups. (g, h) ROC curves at 1, 3 and 5 years for the training and validation groups. (i) Prognostic column line graph. LASSO, least absolute shrinkage and selection operator; ROC, receiver operating characteristic.

Article Snippet: AntiRAN primary antibody (dilution 1:250), anti-CDK1 primary antibody (dilution 1:250), and anti-CDK2 primary antibody (dilution 1:250) were purchased from Proteintech (Wuhan, China) and diluted in phosphatebuffered saline.

Techniques: Biomarker Discovery, Selection

Journal: Archives of oral biology

Article Title: A novel T-cell proliferation-associated gene predicts prognosis and reveals immune infiltration in patients with oral squamous cell carcinoma.

doi: 10.1016/j.archoralbio.2023.105719

Figure Lengend Snippet: Fig. 4. (a-c) Survival analysis based on the T-lymphocyte proliferation regulator signature (RAN, CDK1, CDK2). (d-f) ROC curves of the T-lymphocyte proliferation regulator signature (RAN, CDK1, CDK2) at 1, 3, and 5 years. (g) Risk factor visualisation. (h) Clinical correlation heat map analysis. ROC, receiver operating characteristic.

Article Snippet: AntiRAN primary antibody (dilution 1:250), anti-CDK1 primary antibody (dilution 1:250), and anti-CDK2 primary antibody (dilution 1:250) were purchased from Proteintech (Wuhan, China) and diluted in phosphatebuffered saline.

Techniques:

Journal: Archives of oral biology

Article Title: A novel T-cell proliferation-associated gene predicts prognosis and reveals immune infiltration in patients with oral squamous cell carcinoma.

doi: 10.1016/j.archoralbio.2023.105719

Figure Lengend Snippet: Fig. 5. Immune infiltration analysis (a-c) Infiltration analysis of the T-lymphocyte proliferation regulator signature (RAN, CDK1, CDK2) in 24 immune cell sub populations. (d) Enrichment fraction of each infiltrating cell type expressed between high-risk and low-risk patients. (e) Differences in the StromalScore, Immu neScore, and ESTIMATEScore between high-risk and low-risk patients. *p < 0.05; * *p < 0.01; * **p < 0.001; ns, not statistically significant. (f) Differences in the proportion of PD-1/PD-L1 in high-risk and low-risk patients.

Article Snippet: AntiRAN primary antibody (dilution 1:250), anti-CDK1 primary antibody (dilution 1:250), and anti-CDK2 primary antibody (dilution 1:250) were purchased from Proteintech (Wuhan, China) and diluted in phosphatebuffered saline.

Techniques:

Journal: Archives of oral biology

Article Title: A novel T-cell proliferation-associated gene predicts prognosis and reveals immune infiltration in patients with oral squamous cell carcinoma.

doi: 10.1016/j.archoralbio.2023.105719

Figure Lengend Snippet: Fig. 6. T-lymphocyte proliferation regulator signature expression in OSCC TME-associated cells. (a) Annotation of all cell types in GSE103322 and the percentage of each cell type. (b) Percentages and expression levels of RAN, CDK1, and CDK2. OSCC, oral squamous cell carcinoma; TME, tumour microenvironment.

Article Snippet: AntiRAN primary antibody (dilution 1:250), anti-CDK1 primary antibody (dilution 1:250), and anti-CDK2 primary antibody (dilution 1:250) were purchased from Proteintech (Wuhan, China) and diluted in phosphatebuffered saline.

Techniques: Expressing

Journal: Archives of oral biology

Article Title: A novel T-cell proliferation-associated gene predicts prognosis and reveals immune infiltration in patients with oral squamous cell carcinoma.

doi: 10.1016/j.archoralbio.2023.105719

Figure Lengend Snippet: Fig. 8. T-lymphocyte proliferation regulator signature (RAN, CDK1, and CDK2) combined with the Human Protein Atlas database and immunohistochemical assays to analyze the protein expression levels. Scale bar = 50 µm.

Article Snippet: AntiRAN primary antibody (dilution 1:250), anti-CDK1 primary antibody (dilution 1:250), and anti-CDK2 primary antibody (dilution 1:250) were purchased from Proteintech (Wuhan, China) and diluted in phosphatebuffered saline.

Techniques: Immunohistochemical staining, Expressing

Journal: Archives of oral biology

Article Title: A novel T-cell proliferation-associated gene predicts prognosis and reveals immune infiltration in patients with oral squamous cell carcinoma.

doi: 10.1016/j.archoralbio.2023.105719

Figure Lengend Snippet: Fig. 7. Drug-sensitivity analysis of the T-lymphocyte proliferation regulator signature (RAN, CDK1, and CDK2).

Article Snippet: AntiRAN primary antibody (dilution 1:250), anti-CDK1 primary antibody (dilution 1:250), and anti-CDK2 primary antibody (dilution 1:250) were purchased from Proteintech (Wuhan, China) and diluted in phosphatebuffered saline.

Techniques:

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: NT157 inhibits cell proliferation and sensitizes glioma cells to TRAIL-induced apoptosis by up-regulating DR5 expression.

doi: 10.1016/j.biopha.2022.113502

Figure Lengend Snippet: Fig. 8. Proposed signal pathways. NT157 caused ROS generation and DNA damage, which activated p21 and p27, and subsequently lunched S-phase and M-phase cell cycle arrest through regulating cyclin A1, CDK2, cyclin B1 and CDK1. NT157 also dysfunctioned MAPKs, PI3K/AKT and EGFR-STAT3 pathways, and enhanced TRAIL-induced glioma cells apoptosis by up-regulating DR5 expression.

Article Snippet: CyclinA (sc-271682), anti-p21(sc-6246), CDK1 (sc-53219) antibodies were purchased from Santa Cruz Biotechnology (Shanghai, China). β-actin antibody and IgG were bought from Beijing zhongshan Jinqiao Biotechnology (Beijing, China). p27 (#3686), CDK2 (#18048), Cyclin B1 (#12231), PARP (#9542), Caspase-3 (#9662), PCNA (#13110), Ku70 (#4588), Ku80 (#2753), γ-H2AX (#80312), p-ERK (#4370), ERK (#4695), p-p38 (#4511), p38 (#8690), p-JNK (#9255), JNK (#9252), Akt (#4685), p-Akt (#4060), EGFR (#8083), p-Stat3 (#9145), Stat3 (#9139), DR5(#69400), DR4(#42533) were all purchased from Cell Signaling Technology (CST, USA).

Techniques: Expressing

Journal: Molecular Therapy

Article Title: CDK1-loaded extracellular vesicles promote cell cycle to reverse impaired wound healing in diabetic obese mice

doi: 10.1016/j.ymthe.2025.01.039

Figure Lengend Snippet: Characterization of CDK1-loaded sEVs (A) Schematic of engineering CDK1-loaded sEVs. (B) Determination of EV concentration of mock (empty vector) and CDK1-loaded sEVs ( n = 7 each). (C) Size distribution of sEVs ( n = 7). (D) Representative vFC analyses of sEVs ( n = 7). MFI, mean fluorescent intensity. (E) Immunoblotting of sEVs using EV markers, CDK1 and calnexin. (F) Normalized CDK1 expression based on immunoblotting, region of interest values shown ( n = 4; ∗∗ p < 0.01). (G) Immunoblotting of sEVs using phosphorylation of CDK1 Tyr15 vs. pan-CDK1, and Alix as EV marker.

Article Snippet: Primers designed by TAKARA tools ( https://www.takarabio.com/learning-centers/cloning/primer-design-and-other-tools ) and amplified CDK1 genes from cDNA (catalog no. SC111605, Origene) encoding human CDK1 (NM_001786).

Techniques: Concentration Assay, Plasmid Preparation, Western Blot, Expressing, Phospho-proteomics, Marker

Journal: Molecular Therapy

Article Title: CDK1-loaded extracellular vesicles promote cell cycle to reverse impaired wound healing in diabetic obese mice

doi: 10.1016/j.ymthe.2025.01.039

Figure Lengend Snippet: Testing of the activity of CDK1-loaded sEVs in impaired wound healing (A) Schematic of CDK1-loaded sEVs used in the single-dose treatment of the wound bed of diabetic obese mice. (B) Representative images of wound bed following topical treatment with PBS, mock sEVs, or CDK1-loaded sEVs. (C) Quantification of wound-closure kinetics ( n = 6 per group; ∗ p < 0.05; ∗∗∗∗ p < 0.0001). (D) Representative H&E-stained section of wounds collected on day 3 post-treatment with sEVs (top row: low magnification; bottom row: high magnification). (E) Quantification of epithelial thickness based on imaging analysis of H&E-stained sections ( n = 6; ∗∗∗∗ p < 0.0001). (F) Localization of Ki67 + cells by immunohistochemistry on day 3 post-treatment with sEVs (top row: low magnification; bottom row: high magnification; brown arrows indicate Ki67 + staining). (G) Quantification of Ki67 + cells shows the number of Ki67 + cells per unit area ( n = 6; ∗∗ p < 0.01; ∗∗∗ p < 0.001,).

Article Snippet: Primers designed by TAKARA tools ( https://www.takarabio.com/learning-centers/cloning/primer-design-and-other-tools ) and amplified CDK1 genes from cDNA (catalog no. SC111605, Origene) encoding human CDK1 (NM_001786).

Techniques: Activity Assay, Staining, Imaging, Immunohistochemistry

Journal: Molecular Therapy

Article Title: CDK1-loaded extracellular vesicles promote cell cycle to reverse impaired wound healing in diabetic obese mice

doi: 10.1016/j.ymthe.2025.01.039

Figure Lengend Snippet: Testing the activity of CDK1-loaded sEVs upon human keratinocytes in vitro (A) Immunofluorescence staining of cells with an anti-CDK1 antibody (red), and counterstained with a nuclear stain (blue) post-sEV treatment (scale bar: 50 μm). (B) Proliferation of human keratinocyte following EV treatment using CCK-8 assay ( n = 10; ∗∗ p < 0.01; ∗∗∗∗ p < 0.0001). (C) Representative imaging of in vitro scratch assay in the presence of the proliferation inhibitor mitomycin C following treatment with sEVs and controls (scale bar: 200 μm). (D) Quantification of closure kinetics ( n = 4; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001). (E) Effect of sEV treatment on cell cycle using a cell-permeable DNA dye and analysis by flow cytometry. (F) Quantification of G2,M phase from sEV-treated cells ( n = 3, ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001).

Article Snippet: Primers designed by TAKARA tools ( https://www.takarabio.com/learning-centers/cloning/primer-design-and-other-tools ) and amplified CDK1 genes from cDNA (catalog no. SC111605, Origene) encoding human CDK1 (NM_001786).

Techniques: Activity Assay, In Vitro, Immunofluorescence, Staining, CCK-8 Assay, Imaging, Wound Healing Assay, Flow Cytometry

Journal: Molecular Therapy

Article Title: CDK1-loaded extracellular vesicles promote cell cycle to reverse impaired wound healing in diabetic obese mice

doi: 10.1016/j.ymthe.2025.01.039

Figure Lengend Snippet: Downstream signaling mediated by treatment with CDK1-loaded sEVs in human keratinocytes (A and B) (A) Immunofluorescent localization of p-AKT Ser473 and (B) quantification. (C and D) (C) Localization of p-ERK Thr202/Tyr204 and (D) quantification. (E and F) (E) Localization of phospho-4E-BP1 Thr37/46 and (F) quantification (scale bar: 50 μm) ( n = 8; ∗ p < 0.05; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001). (G–I) Immunoblotting for (G) pan-AKT and p-AKT Ser473 , (H) pan-ERK and phospho-ERK Thr202/Tyr204 , and (I) pan-4E-BP1 and phospho-4E-BP1 Thr37/46 and levels normalized to β-actin.

Article Snippet: Primers designed by TAKARA tools ( https://www.takarabio.com/learning-centers/cloning/primer-design-and-other-tools ) and amplified CDK1 genes from cDNA (catalog no. SC111605, Origene) encoding human CDK1 (NM_001786).

Techniques: Western Blot

Journal: Molecular Therapy

Article Title: CDK1-loaded extracellular vesicles promote cell cycle to reverse impaired wound healing in diabetic obese mice

doi: 10.1016/j.ymthe.2025.01.039

Figure Lengend Snippet: Analysis of histone phosphorylation by following treatment with CDK1-loaded sEVs onto human keratinocytes (A) Immunofluorescent imaging to localize p-Histone H3 Ser10 (red), and counterstained with acetyl-α-tubulin (green) and DAPI for nuclei (blue) following sEV treatment (top row: low magnification; bottom row: high magnification) (scale bars: 50 and 20 μm). (B) Quantification of p-Histone H3 Ser10 following EV treatment ( n = 8; ∗∗∗∗ p < 0.0001). (C) Representative immunofluorescent images of cell-cycle progression following sEV (scale bar: 20 μm). (D) Distribution of mitotic phases based on each sEV treatment ( n = 50 for each treatment).

Article Snippet: Primers designed by TAKARA tools ( https://www.takarabio.com/learning-centers/cloning/primer-design-and-other-tools ) and amplified CDK1 genes from cDNA (catalog no. SC111605, Origene) encoding human CDK1 (NM_001786).

Techniques: Phospho-proteomics, Imaging

Journal: Molecular Therapy

Article Title: CDK1-loaded extracellular vesicles promote cell cycle to reverse impaired wound healing in diabetic obese mice

doi: 10.1016/j.ymthe.2025.01.039

Figure Lengend Snippet: Model for driving entry into the mitotic cycle by treatment of cells with CDK1-loaded sEVs that promote cytosolic signaling and phosphorylation of nuclear histones on chromatin

Article Snippet: Primers designed by TAKARA tools ( https://www.takarabio.com/learning-centers/cloning/primer-design-and-other-tools ) and amplified CDK1 genes from cDNA (catalog no. SC111605, Origene) encoding human CDK1 (NM_001786).

Techniques: Phospho-proteomics

Journal: PLOS ONE

Article Title: Transcriptome-wide analysis of the differences between MCF7 cells cultured in DMEM or αMEM

doi: 10.1371/journal.pone.0298262

Figure Lengend Snippet: Sequences of the primers used in the quantitative real-time polymerase chain reactions.

Article Snippet: The membranes were blocked with TBST (0.01 M Tris-buffered saline (TBS) with 0.1% Tween-20, pH 7.4) containing 5% non-fat dried milk for 1 h and incubated overnight at 4°C with antibodies against GAPDH (RRID: AB_2801390, CW0100M, 1:2000; CWBio, Jiangsu, China), P21 (RRID: AB_10860537, ab109520, 1:500, abcam, Shanghai, China), CDK1 (RRID: AB_11218160, AM06438SU-N, 1:100; Origene, Wuxi, China), and β-actin (RRID: AB_2665433, CW0096M, 1:1000; CWBio, Jiangsu, China).

Techniques:

Journal: PLOS ONE

Article Title: Transcriptome-wide analysis of the differences between MCF7 cells cultured in DMEM or αMEM

doi: 10.1371/journal.pone.0298262

Figure Lengend Snippet: List of differentially expressed genes in MCF7 cells cultured in αMEM vs. DMEM.

Article Snippet: The membranes were blocked with TBST (0.01 M Tris-buffered saline (TBS) with 0.1% Tween-20, pH 7.4) containing 5% non-fat dried milk for 1 h and incubated overnight at 4°C with antibodies against GAPDH (RRID: AB_2801390, CW0100M, 1:2000; CWBio, Jiangsu, China), P21 (RRID: AB_10860537, ab109520, 1:500, abcam, Shanghai, China), CDK1 (RRID: AB_11218160, AM06438SU-N, 1:100; Origene, Wuxi, China), and β-actin (RRID: AB_2665433, CW0096M, 1:1000; CWBio, Jiangsu, China).

Techniques: Cell Culture, Expressing

Journal: bioRxiv

Article Title: Cdk activity drives senescence from G2 phase

doi: 10.1101/041723

Figure Lengend Snippet: ( A ) Mean and standard deviation from 4 independent experiments of RPE cells treated with Etoposide and after 1h with Roscovitine, MK-1775 (MK) or with DMSO. Alternatively cells were transfected with RNAi for Cdk1 and Cdk2 at 24 and 48h before damage induction in 3 independent experiments. Cells were stained for β-Galactosidase 4 days later. Statistical hypothesis testing was performed using two-sided t -test. (B) Quantification of nuclear H3K9Me2, HP1b, and IL-6 levels in RPE cells treated with Etoposide and after 1h with Roscovitine, MK-1775 (MK) or with DMSO. Alternatively cells were transfected with RNAi for Cdk1 and Cdk2 at 24 and 48h before damage induction. Cells were fixed 5 days after damage induction. Statistical hypothesis testing was performed using two-sided t -test. (C) Analysis of proliferative capacity. RPE cells were treated with Etoposide and 1h later with Roscovitine, MK-1775 or DMSO. Cells were counted after 5 days, reseeded into fresh medium and counted again after 2 more days. Mean and standard deviation of 3 independent experiments ran in quadruplicates are shown. Statistical hypothesis testing was performed using two-sided t -test. (D) Analysis of clonogenic capacity. RPE cells were treated with Etoposide and 1h later with Roscovitine, MK-1775 or DMSO. After 5 days 5000 cells were reseeded into fresh medium and the number of colonies was assessed one week later. Normalized mean and standard deviation of 3 independent experiments ran in quadruplicates are shown. Statistical hypothesis testing was performed using two-sided t -test.

Article Snippet: The following antibodies were used: Lamin A/C pS22 (1:400; #2026 Cell Signaling), Cdc6 pS54 EPR759Y (1:200; ab75809 abcam), Cyclin B1 pS126 (1:200; ab55184 abcam, 1:100; ab3488 abcam), p53 DO-1 (1:500; sc-126 Santa Cruz), p53 pSer15 (1:200, #9284 Cell Signaling), p21 12D1 (1:1000; #2947 Cell Signaling), β-tubulin 9F3 (1:1000; #2128S Cell Signaling), Cdk1 POH1 (1:1000; #9116 Cell Signaling), Cdk1 (1:200; HPA003387 Atlas antibodies), Cdk2 78B2 (1:1000; #2564 Cell Signaling), Lamin A/C (1:2000; #4777 Cell Signaling), GAPDH (1:15000-25000; G9545 Sigma Aldrich), pKap1 (1:500; A300-767A Bethyl antibodies) and pChk2 (1:1000; #2661 Cell Signaling).

Techniques: Standard Deviation, Transfection, Staining

Journal: bioRxiv

Article Title: Cdk activity drives senescence from G2 phase

doi: 10.1101/041723

Figure Lengend Snippet: (A) Western blot of a typical siRNA knockdown of Cdk1 and Cdk2 in a 96-well format. Cells were transfected with RNAi for Cdk1 and Cdk2 24 and 48h before sample preparation. (B) Increased nuclear size as an indicator of cellular senescence . RPE cells were treated with Etoposide. Roscovitine or DMSO were added 1h later. Alternatively, cells were transfected with siRNA for Cdk1 and Cdk2 at 24 and 48h before damage induction. Cells were fixed 4 days after damage induction, stained with DAPI and nuclear size was assessed in more than 250 cells for each condition. Statistical hypothesis testing was performed using two-sided t -test. (C) Quantification of nuclear H3K9Me2, HP1b, and IL-6 levels in control RPE cells and cells treated with Etoposide for 5 days. Statistical hypothesis testing was performed using two-sided t -test. (D) Quantification of nuclear foci intensity of H3K9Me2 and HP1b, and cytoplasmic IL-6 levels. RPE cells treated with Etoposide and after 1h with Roscovitine, MK-1775 (MK) or with DMSO. Alternatively cells were transfected with siRNA for Cdk1 and Cdk2 at 24 and 48h before damage induction in 3 independent experiments. Cells were fixed 5 days after damage induction. Statistical hypothesis testing was performed using two-sided t -test. ( E )Colony formation capacity of RPE cells treated with Etoposide and DMSO,Roscovitine (Rosc) or MK-1775. Data from 3 independent experiments are shown.

Article Snippet: The following antibodies were used: Lamin A/C pS22 (1:400; #2026 Cell Signaling), Cdc6 pS54 EPR759Y (1:200; ab75809 abcam), Cyclin B1 pS126 (1:200; ab55184 abcam, 1:100; ab3488 abcam), p53 DO-1 (1:500; sc-126 Santa Cruz), p53 pSer15 (1:200, #9284 Cell Signaling), p21 12D1 (1:1000; #2947 Cell Signaling), β-tubulin 9F3 (1:1000; #2128S Cell Signaling), Cdk1 POH1 (1:1000; #9116 Cell Signaling), Cdk1 (1:200; HPA003387 Atlas antibodies), Cdk2 78B2 (1:1000; #2564 Cell Signaling), Lamin A/C (1:2000; #4777 Cell Signaling), GAPDH (1:15000-25000; G9545 Sigma Aldrich), pKap1 (1:500; A300-767A Bethyl antibodies) and pChk2 (1:1000; #2661 Cell Signaling).

Techniques: Western Blot, Knockdown, Transfection, Sample Prep, Staining, Control

Journal: bioRxiv

Article Title: Cdk activity drives senescence from G2 phase

doi: 10.1101/041723

Figure Lengend Snippet: ( A ) Asynchronous growing RPE Cyclin A2-eYFP cells were treated with Etoposide or NCS for 4h, lysed and immunoprecipitated with anti-GFP or control antibody. Kinase assay was performed using GST-Cdk substrate peptide and phosphorylation was detected by autoradiography. Co-immunoprecipitation of Cdk2 and Cdk1 was determined by immunoblotting. Arrowhead shows position of Cdk1, empty arrowhead indicates position of IgG. WCL, whole cell lysate. (B) RPE Cyclin B1-eYFP cells were released for 6h from a thymidine block, treated with Etoposide or NCS for 4h, lysed and immunoprecipitated with anti-GFP or control antibody. Kinase assay was performed using GST-LAMS22 substrate peptide and phosphorylation was detected by antibody against Lamin A/C phosphorylated at Ser22. WCL, whole cell lysate. (C) RPE cells were released for 6h from a thymidine block, treated with Etoposide for 4h, lysed and immunoprecipitated with anti-Cdk2 or control antibody. Kinase assay was performed in the absence or presence of Roscovitine and kinase activity was determined as in (a).

Article Snippet: The following antibodies were used: Lamin A/C pS22 (1:400; #2026 Cell Signaling), Cdc6 pS54 EPR759Y (1:200; ab75809 abcam), Cyclin B1 pS126 (1:200; ab55184 abcam, 1:100; ab3488 abcam), p53 DO-1 (1:500; sc-126 Santa Cruz), p53 pSer15 (1:200, #9284 Cell Signaling), p21 12D1 (1:1000; #2947 Cell Signaling), β-tubulin 9F3 (1:1000; #2128S Cell Signaling), Cdk1 POH1 (1:1000; #9116 Cell Signaling), Cdk1 (1:200; HPA003387 Atlas antibodies), Cdk2 78B2 (1:1000; #2564 Cell Signaling), Lamin A/C (1:2000; #4777 Cell Signaling), GAPDH (1:15000-25000; G9545 Sigma Aldrich), pKap1 (1:500; A300-767A Bethyl antibodies) and pChk2 (1:1000; #2661 Cell Signaling).

Techniques: Immunoprecipitation, Control, Kinase Assay, Phospho-proteomics, Autoradiography, Western Blot, Blocking Assay, Activity Assay

Journal: bioRxiv

Article Title: Cdk activity drives senescence from G2 phase

doi: 10.1101/041723

Figure Lengend Snippet: (A) Representative Western blot of RPE cells treated with Etoposide and with a combination of Roscovitine, RO-3306 and NU6140 (Cdk inh.) or mock treatment with DMSO 1h later. Cell lysates were prepared at the indicated time points. C, control. (B) Representative Western blots of RPE cells treated with Etoposide and with Roscovitine (Cdk1/2), MK1775 (Wee1), SB202190 (p38) or mock treatment with DMSO 1h later (left blot). Alternatively cells were transfected with the indicated siRNA at 24 and 48h before damage induction (right blot). C, Control. (C) Representative Western blots of RPE cells transfected with Cdk1AF-GFP or control, with and without 4h Etoposide treatment. (D) Quantification of nuclear p21 levels and nuclear p53 level versus estimated time. Cells were sorted for DAPI and Cyclin B1. Cells were treated with Etoposide at 1 µM concentration or mock treated with DMSO (control). Roscovitine, a combination of RO-3306 and NU6140, or DMSO was added 1h after Etoposide treatment. Cells were fixed after 4h. More than 350 cells were analyzed for each condition. ( E )Immunofluorescence quantification of nuclear p21 levels in G2 cells. RPE cellswere treated with Etoposide and 1h later with Roscovitine (Cdk inhibition) incombination with the indicated drug (DMSO, CHX, MG, nutlin). Cells were fixed 4hafter damage and G2 cells were identified according to DNA content using DAPIstaining. ( F )Representative Western blot of RPE cells treated with Etoposide at time point 0and 1h later with Cycloheximide alone, or Cycloheximide in combination with Cdkinhibition (Roscovitine, RO-3306 and NU6140 - Cdk inh.). Cell lysates wereprepared at the indicated time points. Below, quantification of p21 and p53degradation kinetics. The average and standard deviation of three independentexperiments are shown. ( G )RPE cells were treated with Etoposide and 1h later with Roscovitine, acombination of RO-3306 and NU6140, or mock treatment with DMSO. Means andstandard deviation of RT-qPCR measurements obtained in 4 independent experimentsare shown. Statistical hypothesis testing was performed using two-sided t -test. (H) Cdk activity determines cell fate decisions towards mitosis in unperturbed conditions or towards senescence upon DNA damage.

Article Snippet: The following antibodies were used: Lamin A/C pS22 (1:400; #2026 Cell Signaling), Cdc6 pS54 EPR759Y (1:200; ab75809 abcam), Cyclin B1 pS126 (1:200; ab55184 abcam, 1:100; ab3488 abcam), p53 DO-1 (1:500; sc-126 Santa Cruz), p53 pSer15 (1:200, #9284 Cell Signaling), p21 12D1 (1:1000; #2947 Cell Signaling), β-tubulin 9F3 (1:1000; #2128S Cell Signaling), Cdk1 POH1 (1:1000; #9116 Cell Signaling), Cdk1 (1:200; HPA003387 Atlas antibodies), Cdk2 78B2 (1:1000; #2564 Cell Signaling), Lamin A/C (1:2000; #4777 Cell Signaling), GAPDH (1:15000-25000; G9545 Sigma Aldrich), pKap1 (1:500; A300-767A Bethyl antibodies) and pChk2 (1:1000; #2661 Cell Signaling).

Techniques: Western Blot, Control, Transfection, Concentration Assay, Immunofluorescence, Inhibition, Standard Deviation, Quantitative RT-PCR, Activity Assay