Journal: Journal of Inflammation Research

Article Title: Sodium Propionate Alleviates Atopic Dermatitis by Inhibiting Ferroptosis via Activation of LTBP2/FABP4 Signaling Pathway

doi: 10.2147/JIR.S495271

Figure Lengend Snippet: SP improves genes associated with inflammation and skin growth in AD cell models. ( A and B ) The cell viability rate and cytotoxicity of AD cell models after SP intervention. ( C – F ) Expression of related proteins of LTBP2, FABP4, CDK1, LOR and FLG. ( G – M ) mRNA expression of LOR, FLG, TSLP, TNF-α, IL-4,IL-13, and cxcl1. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: The CDK1, FABP4, and LOR antibodies were obtained from MedChemExpress (Shanghai, China), the LTBP2 antibody was sourced from Biodragon (Suzhou, China), the FLG antibody was acquired from ABclonal (Wuhan, China), and the GAPDH antibody was obtained from ProteinTech (Wuhan, China).

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

Journal: Cell reports

Article Title: Hypoxia drives HIF2-dependent reversible macrophage cell cycle entry.

doi: 10.1016/j.celrep.2024.114471

Figure Lengend Snippet: Figure 2. Hypoxia (1% oxygen) increases susceptibility to virus infection due to cell cycle entry and SAMHD1 deactivation (A) Fluorescence images of HIV-1 and SIVmac infection with Hoechst for nuclei and GFP for virus- infected cells in both normoxia and hypoxia. The number of GFP-positive cells was quantitated and normalized to the total number of cells to give a single-round infection rate. The rates were then normalized with those in normoxia conditions for either HIV-1 or SIVmac infection. Duplicate in- fections per donor are plotted for each donor in each condition (n = 3–4). One-sample t test was used for statistical analyses, with error bars rep- resenting SEM and *p < 0.5. The scale bar repre- sents 100 mm. (B) Representative western blots for conditions in (A). (C) Western blots of MDM lysates probing for CDK1, MCM2, SAMHD1, and pSAMHD1 in hyp- oxia are shown on the left. Shown on the right is infectivity of VSV-G pseudotyped HIV-1 under 1% oxygen tension and SAMHD1 knockdown. The number of GFP-positive cells was quantitated us- ing microscopy. One representative experiment from two is shown, with error bars representing SEM from technical duplicates of one experiment. For western blots, the antibodies used for blotting are shown on the right of the blots with protein markers on the left.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse anti human HIF1a BD 610958 Rabbit anti human HIF2a Cell Signaling 7096S Rabbit anti HIF1a Cell Signaling 36169 Mouse anti MCM2 BD 610700 Rabbit anti SAMHD1 Proteintech 12586-1-AP Rabbit anti pSAMHD1(Thr592) Cell Signaling 89930S Mouse anti b-actin Abcam ab6276 Rabbit anti human CDK1 Bethyl A303-664A Rabbit anti pERK1/2 Cell Signaling 4370T Rat anti ERK1/2 BioLegend 686902 NCL-L-Geminin Leica N/A Biological samples Apheresis cones NHS Blood and Transplant N/A Chemicals, peptides, and recombinant proteins PT2385 Medchemexpress HY-12867 Daprodustat Medchemexpress HY-17608 Palbociclib (HY-A0065/CS-3110) Sigma PZ0383 U0126 Calbiochem 662005 Tak632 Selleckchem S7291 AZ-628 APExBIO A8371 AB Human Serum Sigma H4522 FBS Thermofisher A5256701 Fast SYRB green master mix Thermofisher 4385610 PhosSTOP Merck 4906845001 Fugene HD transfection reagent Promega E2311 RNAiMAX Transfection Reagent Thermofisher 13778075 Purelink RNA mini kit Thermofisher 12183018A 10x Cell lysis buffer Cell Signaling 9803 Lymphoprep Stemcelltechnologies 07851 MCSF Tonbo Biosciences 21-8789-U010 Critical commercial assays Blot 4–12% Bis-Tris Protein Gel Thermofisher NW04120BOX pHrodo E.coli BioParticles Thermofisher P3561 SuperScript III First-Strand Synthesis System Thermofisher 18080051 ECL Prime Western Blotting Detection Reagent Sigma RPN2232 Click-it Edu kit Thermofisher C10340 RNeasy Mini Kit Qiagen 74104 PureLink RNA extraction Kit Thermofisher 12183018A SMARTer Stranded Total RNA-Seq v2 - Pico Input Mammalian kit Takara 634411 Deposited data Transcriptomic data from MDM in hypoxia vs. normoxia GSE269699 Experimental models: Cell lines HEK-293T G. Towers N/A (Continued on next page) 16 Cell Reports 43, 114471, July 23, 2024

Techniques: Virus, Infection, Fluorescence, Western Blot, Knockdown, Microscopy

Journal: Cell reports

Article Title: Hypoxia drives HIF2-dependent reversible macrophage cell cycle entry.

doi: 10.1016/j.celrep.2024.114471

Figure Lengend Snippet: Figure 3. The RAS/RAF/MEK/ERK pathway is involved in hypoxia-induced macrophage cell cycle entry (A) A diagram showing the MEK/ERK pathway and the specific inhibitors of different targets within this pathway. Targets with increased levels of mRNA expression (Figure S2) in hypoxic MDMs are highlighted in blue. (B) Abrogation of CDK1 expression induced by inhibitors of RAF, MEK, and CDK4/6, verifying drug specificity targeting the RAS/RAF/MEK/ERK pathway in MDMs under normoxic conditions. In- hibitors used were U0126 (10 mM), palbociclib (pal; 1 mM), Tak632 (Tak; 2 mM), and AZ628 (AZ; 1 mM). Shown is a representative western blot of cell ly- sates harvested post 2 days of incubation under hypoxia or with addition of the inhibitors under normoxia. (C) Left: Representative western blot of lysates from cells treated with U0126 (10 mM) and Pal (10 mM) or left untreated, followed by pseudotyped HIV-1 infection in hypoxia. Right: The number of GFP-positive cells was quantitated and normal- ized to the total number of cells to give a single- round infection percentage. The percentage of infection with U0126 or Pal was then normalized to untreated. Duplicate infections are plotted for each donor in each condition, with error bars repre- senting SEM (n = 2–3). One-sample t test was used for statistical analyses; ***p < 0.01. For western blots, the antibodies used for blotting are shown on the right of the blots, with protein markers on the left.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse anti human HIF1a BD 610958 Rabbit anti human HIF2a Cell Signaling 7096S Rabbit anti HIF1a Cell Signaling 36169 Mouse anti MCM2 BD 610700 Rabbit anti SAMHD1 Proteintech 12586-1-AP Rabbit anti pSAMHD1(Thr592) Cell Signaling 89930S Mouse anti b-actin Abcam ab6276 Rabbit anti human CDK1 Bethyl A303-664A Rabbit anti pERK1/2 Cell Signaling 4370T Rat anti ERK1/2 BioLegend 686902 NCL-L-Geminin Leica N/A Biological samples Apheresis cones NHS Blood and Transplant N/A Chemicals, peptides, and recombinant proteins PT2385 Medchemexpress HY-12867 Daprodustat Medchemexpress HY-17608 Palbociclib (HY-A0065/CS-3110) Sigma PZ0383 U0126 Calbiochem 662005 Tak632 Selleckchem S7291 AZ-628 APExBIO A8371 AB Human Serum Sigma H4522 FBS Thermofisher A5256701 Fast SYRB green master mix Thermofisher 4385610 PhosSTOP Merck 4906845001 Fugene HD transfection reagent Promega E2311 RNAiMAX Transfection Reagent Thermofisher 13778075 Purelink RNA mini kit Thermofisher 12183018A 10x Cell lysis buffer Cell Signaling 9803 Lymphoprep Stemcelltechnologies 07851 MCSF Tonbo Biosciences 21-8789-U010 Critical commercial assays Blot 4–12% Bis-Tris Protein Gel Thermofisher NW04120BOX pHrodo E.coli BioParticles Thermofisher P3561 SuperScript III First-Strand Synthesis System Thermofisher 18080051 ECL Prime Western Blotting Detection Reagent Sigma RPN2232 Click-it Edu kit Thermofisher C10340 RNeasy Mini Kit Qiagen 74104 PureLink RNA extraction Kit Thermofisher 12183018A SMARTer Stranded Total RNA-Seq v2 - Pico Input Mammalian kit Takara 634411 Deposited data Transcriptomic data from MDM in hypoxia vs. normoxia GSE269699 Experimental models: Cell lines HEK-293T G. Towers N/A (Continued on next page) 16 Cell Reports 43, 114471, July 23, 2024

Techniques: Expressing, Western Blot, Incubation, Infection

Journal: International Journal of Nanomedicine

Article Title: Preparation of coffee oil-algae oil-based nanoemulsions and the study of their inhibition effect on UVA-induced skin damage in mice and melanoma cell growth

doi: 10.2147/ijn.s144705

Figure Lengend Snippet: Figure 7 Effects of coffee oil-algae oil nanoemulsions on protein expressions of cyclin B, CDK2, cyclin A, and CDK1 (A), p53 and p21 (B), and Bax, Bcl-2, cytochrome C (C). Notes: Control cells are incubated with medium only. Results are presented as mean ± standard deviation of triplicate determinations. Data with different letters (A–C) are significantly different at p,0.05.

Article Snippet: The primary antibodies include mouse monoclonal antiα-tubulin antibody (Sigma–Aldrich Co.), mouse anti-cyclin A and rabbit anti-Bax (EMD Millipore), mouse anti-CDK2, mouse anti-cytochrome C, mouse anti-p21, mouse anticyclin B, and mouse anti-Bcl-2 (BD Biosciences), as well as anti-cdc2 (CDK1) and mouse anti-p53 (Novus Biologicals Co., Littleton, CO, USA).

Techniques: Algae, Control, Incubation, Standard Deviation

Journal: Genetics

Article Title: Diploidy confers genomic instability in Schizosaccharomyces pombe

doi: 10.1093/genetics/iyaf078

Figure Lengend Snippet: Depletion of Rad52 and Mad2 using the AID system results in loss of viability in diploids. A, b) Immunoblot and quantification of Mad2 degradation in Mad2-AID haploids and Mad2-AID homozygous diploids. Cells were treated with 100 nM 5′a-IAA and sampled at the indicated time points. Cdc2 was used as the loading control. c) Control and Mad2-AID strains were spotted onto YES plates with and without 100 nM 5′a-IAA and/or 10 µg/mL thiabendazole (TBZ). Plates were incubated at 32°C for 3 days and then imaged. d) Immunoblot and quantification of Rad52 degradation in Rad52-AID homozygous diploids. Conditions were the same as the Mad2 assay. e) Control and Rad52-AID strains were spotted onto YES plates with and without 100 nM 5′a-IAA and/or 2 mM HU. Plates were incubated at 32°C for 3 days and then imaged.

Article Snippet: The following antibodies and dilutions were used for immunoblotting: α- cdc2 (Novus Biologicals NB100-2716; 1:2,000), α-V5 (Abcam ab27671; 1:1,000), α-myc (Abcam ab9106 and Novus Biologicals NBP2-52636; 1:1,000), α-Alpha Tubulin (Millipore Sigma T5168; 1:1,000), α-Rabbit Alexa Fluor 488 (Thermo Scientific A32790; 1:1,000), and α-Mouse Alexa Fluor 488 (Thermo Scientific A28175; 1:1,000).

Techniques: Western Blot, Control, Incubation