cdc25c  (Cell Signaling Technology Inc)

Journal: Journal of Advanced Research

Article Title: Tankyrase activity is essential for asymmetric division and chromosome segregation in oocyte meiosis

doi: 10.1016/j.jare.2025.07.008

Figure Lengend Snippet: TNKS regulates BubR1/MPF activity, and Securin accumulation in mouse oocyte. (A) Representative images of BubR1 signals at the kinetochore in oocytes from the control and JW55 treatment groups. Green, BubR1. Red, ACA. Blue, DNA. Bar = 5 μm. (B) The percentage of positive BubR1 signals at the kinetochore in oocytes from the control (n = 53) and JW55 (n = 50) treatment groups. ***, P < 0.001. (C) Representative images of oocytes PB1 extrusion after 12 h culture in the Control, control + Milri, control + 10 μM ETOP, JW55 + 10 μM ETOP and JW55 groups. GV oocytes were exposed to etoposide (ETOP) at concentrations of 1 μM and 10 μM for 30 min in the presence 4 μM milrinone (Milri) to prevent GVBD during this treatment. Bar = 80 μm. (D) The percentage of PB1 or large PB1 extrusion in the control (n = 94), control + Milri (n = 89), control + 1 μM ETOP (n = 98), control + 10 μM ETOP (n = 97), JW55 + 1 μ M ETOP (n = 128), JW55 + 10 μM ETOP (n = 120), and JW55 treatment (n = 114) oocytes after ETOP pretreatment. ns, P > 0.05. **, P < 0.01. ***, P < 0.001. (E) Representative images of γ-H2AX in MI stage oocyte from the control + 10 μM ETOP and JW55 + 10 μM ETOP oocytes. Red, γ-H2AX. Blue, DNA. Bar = 5 μm. (F) Relative intensity of γ-H2AX in MI stage oocyte from the control + 10 μM ETOP (n = 47) and JW55 + 10 μM ETOP (n = 39) oocytes. ***, P < 0.001. (G) Western blot of Cyclin B1 and Securin in oocytes from the control and JW55 treatment groups after 6 h of culture. (H) Western blot of pT161-CDK1 and CDC20 in oocytes from the control and JW55 treatment groups after 6 h of culture, and band intensity analysis of their protein levels in oocytes from the control and JW55 treatment groups. *, P < 0.05. **, P < 0.01. (I) Band intensity analysis of pT161-CDK1, pY15-CDK1, and Securin in oocytes from the control and JW55 treatment groups after 3 h of culture. ns, P > 0.05. *, P < 0.05. (J) Band intensity analysis of CDC25C, Cyclin B1, and pT14-CDK1 in oocytes from the control and JW55 treatment groups after 3 h of culture. *, P < 0.05. **, P < 0.01. (K) Protein-protein interaction network analysis of cell cycle-related proteins identified by mass spectrometry and MPF/Securin/CDC20/CDC25C upon STRING database. Different node color indicated core and non-core nodes, while edge thickness edge represented interaction strength. (L) Co-IP analysis with an anti-TNKS antibody. The immunoblot was probed with anti-CDC20 and anti-CDC25C antibodies. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Rabbit anti-α-Tubulin antibody (11224-1-AP), rabbit polyclonal anti-GAPDH antibody (10494-1-AP), rabbit anti-GRP78 antibody (11587-1-AP), rabbit anti-TNKS1 antibody (18030-1-AP), rabbit anti-Ran antibody (10469-1-AP), and rabbit anti-Rab11a antibody (67902-1-Ig), rabbit anti-Securin antibody (18040-1-AP), rabbit anti-CDC25C antibody (66912-1-Ig), rabbit anti-Formin2 antibody (11259-1-AP), rabbit anti-BubR1 (11504-2-AP) were from Proteintech.

Techniques: Activity Assay, Control, Western Blot, Mass Spectrometry, Co-Immunoprecipitation Assay

Journal: Drug Design, Development and Therapy

Article Title: The Effect of AZD5153 on Radiosensitivity in Pancreatic Cancer Cells Through ATM-chk1 Pathway

doi: 10.2147/DDDT.S568551

Figure Lengend Snippet: Effect of AZD5153 on protein expression in irradiated pancreatic cancer cells, n=6. ( a and b ) Western blot analysis was performed to detect the expression of BRD4, c-Myc in different groups, β-actin was used as a loading control. ( c and d ) The protein expression of p-ATM, ATM, p-chk1, chk1, p-cdc25, cdc25, p-cdc2, cdc2 in different groups was detected by western blot, β-actin was used as a loading control. Results shown are the means ± SD of 3 independent experiments with similar results for all assays. Significance was determined by Student’s t -test (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: The membranes were blocked with 5% skim milk for 2 h, and incubated with primary antibodies against BRD4 (Cell Signaling Technology, Cat# 13440s), c-Myc (Cell Signaling Technology, Cat# 18583s), p-ATM (Abcam, Cat# ab81292), ATM (Cell Signaling Technology, Cat# 2873s), p-cdc25C (Cell Signaling Technology, Cat# 4901s), cdc25C (Cell Signaling Technology, Cat# 4688s), p-chk1 (Cell Signaling Technology, Cat# 2348s), chk1 (Abcam, Cat# ab40866), p-cdc2 (Cell Signaling Technology, Cat# 4539s), cdc2 (Cell Signaling Technology, Cat# 9116s), γ-H2AX (Cell Signaling Technology, Cat# 9718s), cleaved PARP (Cell Signaling Technology, Cat# 5625s), Bax (Cell Signaling Technology, Cat# 14796s), β-actin (Abcam, Cat# ab6276),Vinculin (Cell Signaling Technology, Cat# 13901s) overnight at 4 °C.

Techniques: Expressing, Irradiation, Western Blot, Control

Journal: Drug Design, Development and Therapy

Article Title: The Effect of AZD5153 on Radiosensitivity in Pancreatic Cancer Cells Through ATM-chk1 Pathway

doi: 10.2147/DDDT.S568551

Figure Lengend Snippet: The effect of overexpression of chk1 on enhancing the radiosensitivity of AZD5153, n=6. ( a ) Overexpression of chk1 protein in Capan2 cells by chk1 plasmid; ( b ) Quantitative statistics of apoptosis in Capan2 cells caused by chk1 overexpression; ( c ) Representative results of the effect of chk1 overexpression on apoptosis of Capan2 cells. Results shown are the means ± SD of 3 independent experiments with similar results for all assays. Significance was determined by Student’s t -test (* p < 0.05, *** p < 0.001).

Article Snippet: The membranes were blocked with 5% skim milk for 2 h, and incubated with primary antibodies against BRD4 (Cell Signaling Technology, Cat# 13440s), c-Myc (Cell Signaling Technology, Cat# 18583s), p-ATM (Abcam, Cat# ab81292), ATM (Cell Signaling Technology, Cat# 2873s), p-cdc25C (Cell Signaling Technology, Cat# 4901s), cdc25C (Cell Signaling Technology, Cat# 4688s), p-chk1 (Cell Signaling Technology, Cat# 2348s), chk1 (Abcam, Cat# ab40866), p-cdc2 (Cell Signaling Technology, Cat# 4539s), cdc2 (Cell Signaling Technology, Cat# 9116s), γ-H2AX (Cell Signaling Technology, Cat# 9718s), cleaved PARP (Cell Signaling Technology, Cat# 5625s), Bax (Cell Signaling Technology, Cat# 14796s), β-actin (Abcam, Cat# ab6276),Vinculin (Cell Signaling Technology, Cat# 13901s) overnight at 4 °C.

Techniques: Over Expression, Plasmid Preparation

Journal: Journal of Advanced Research

Article Title: Multi-omics approach reveals TGF-β signaling-driven senescence in periodontium stem cells

doi: 10.1016/j.jare.2024.12.037

Figure Lengend Snippet: TGF-β1-induced ROS accumulation leads to G2 arrest through ATM signaling. (A). RT-qPCR analysis of ATM signaling in TGF-β1 and/or its inhibitor-treated PDLSCs; (B). Western blot analysis of ATM signaling in TGF-β1 and/or its inhibitor-treated PDLSCs; (C). Western blot analysis of ATM signaling phosphorylated activation in the condition of ATM or CHK2 knockdown; (D). Western blot analysis of ATM signaling phosphorylated activation under N-acetyl-l-cysteine (NAC) treatment; (E). Western blot analysis of ATM signaling phosphorylated activation under decitabine treatment. Reagent doses and duration: TGF-β1 (10 ng/ml, 48 h), NAC (4 mM, 48 h), decitabine (10uM, 48 h). Predicted molecular weight: ATM 350 kDa, pATM 370 kDa, CDC25C&pCDC25C 60 kDa, CHK1&pCHK1 54 kDa, CHK2&pCHK2 62 kDa, β-Tubulin 55 kDa; GAPDH 37 kDa. Statistical significance: * p < 0.05, ** p < 0.01, *** p < 0.001, ns denotes not significant by one-way analysis of variance followed by the Tukey’s test. Error bars represent means ± SEM of at least three independent experiments.

Article Snippet: Antibodies used in this study were listed as follows: antibodies from Abmart: anti-β-Tubulin ( M20005 ); antibodies from Huabio: anti-GAPDH recombinant rabbit monoclonal antibody (ET1601-4), anti-DNMT1 recombinant rabbit monoclonal antibody (ET1702-77), anti-DNMT3A recombinant rabbit monoclonal antibody (ET1609-31), anti-DNMT3B recombinant rabbit monoclonal antibody (ET1605-9), anti-p16 INK4A recombinant rabbit monoclonal antibody (ET1608-62), anti-AMPKγ1 recombinant mouse monoclonal antibody (EM2001-06), anti-AMPK alpha 1 recombinant rabbit monoclonal antibody (ET1608-40), anti-phospho-AMPK alpha 1 (S496) recombinant rabbit monoclonal antibody (ET1612-72), HRP-conjugated goat anti-rabbit IgG goat polyclonal antibody (HA1001), HRP-conjugated goat anti-mouse IgG polyclonal antibody (HA1006); antibodies from Abcam: anti-ATM antibody (ab32420), anti-phospho-ATM (S1981) antibody (ab81292), anti-CHK1 antibody (ab40866), anti-phospho-CHK1 (S296) antibody (ab79758), anti-CDC25C antibody (ab32444), anti-p21 antibody (ab109199), anti-CHK2 antibody (ab109413), anti-histone H3 (tri-methyl K9) (ab176916), anti-phospho-γ-H2AX (S139) antibody (ab81299), anti-HMGB1 antibody (ab79823); antibodies from CST: anti-Ki67 (D3B5) rabbit mAb (#9129), anti-phospho-CHK2 (Thr68) rabbit mAb (#2197), anti-phospho-CDC25C (Ser216) rabbit mAb (#4901); antibodies from Bioss: goat anti-rabbit IgG antibody (H + L), FITC-conjugated (bs-0295G-FITC); goat anti-rat IgG antibody (H + L), cyanine 3-conjugated (bs-0293G-Cy3); antibodies from Biolegend: APC anti-human CD34 antibody (#343509), FITC anti-human CD146 antibody (#361011).

Techniques: Quantitative RT-PCR, Western Blot, Activation Assay, Knockdown, Molecular Weight

Journal: Journal of Advanced Research

Article Title: Multi-omics approach reveals TGF-β signaling-driven senescence in periodontium stem cells

doi: 10.1016/j.jare.2024.12.037

Figure Lengend Snippet: Proposed model for how TGF-β induces G2 arrest-related senescence in PDLSCs. TGF-β signaling induces irreversible G2 arrest in PDLSCs through the ATM signaling pathway in response to ROS accumulation and DNA damage. Specifically, TGF-β1-induced DNMTs expression, including DNMT1, DNMT3A, and DNMT3B, triggers the CpG sites hypermethylation in 5′UTR of PRKAG2 , encoding AMPKγ2, followed by its declined translation and ROS accumulation; meanwhile, NOX4 serves as another source of excessive ROS independent of DNA methylation. Overburdened ROS leads to DNA damage through the activation of the ATM-CHK-CDC25C axis. Of note, the phosphorylation of CDC25C in Ser216 impedes PDLSCs from entering into mitosis, disrupting the G2/M transition. Eventually, constant ROS stress and accumulated DNA damage impel the cell cycle to permanent stagnation, namely cellular senescence. Other well-established senescence-related proteins, including p16 and p21, may participate in maintaining G2 cell cycle arrest, awaiting further validation. Overall, our study demonstrates that TGF-β1 induces G2 arrest-related senescence in PDLSCs primarily due to epigenetic regulation, excessive ROS generation, and DNA damage.

Article Snippet: Antibodies used in this study were listed as follows: antibodies from Abmart: anti-β-Tubulin ( M20005 ); antibodies from Huabio: anti-GAPDH recombinant rabbit monoclonal antibody (ET1601-4), anti-DNMT1 recombinant rabbit monoclonal antibody (ET1702-77), anti-DNMT3A recombinant rabbit monoclonal antibody (ET1609-31), anti-DNMT3B recombinant rabbit monoclonal antibody (ET1605-9), anti-p16 INK4A recombinant rabbit monoclonal antibody (ET1608-62), anti-AMPKγ1 recombinant mouse monoclonal antibody (EM2001-06), anti-AMPK alpha 1 recombinant rabbit monoclonal antibody (ET1608-40), anti-phospho-AMPK alpha 1 (S496) recombinant rabbit monoclonal antibody (ET1612-72), HRP-conjugated goat anti-rabbit IgG goat polyclonal antibody (HA1001), HRP-conjugated goat anti-mouse IgG polyclonal antibody (HA1006); antibodies from Abcam: anti-ATM antibody (ab32420), anti-phospho-ATM (S1981) antibody (ab81292), anti-CHK1 antibody (ab40866), anti-phospho-CHK1 (S296) antibody (ab79758), anti-CDC25C antibody (ab32444), anti-p21 antibody (ab109199), anti-CHK2 antibody (ab109413), anti-histone H3 (tri-methyl K9) (ab176916), anti-phospho-γ-H2AX (S139) antibody (ab81299), anti-HMGB1 antibody (ab79823); antibodies from CST: anti-Ki67 (D3B5) rabbit mAb (#9129), anti-phospho-CHK2 (Thr68) rabbit mAb (#2197), anti-phospho-CDC25C (Ser216) rabbit mAb (#4901); antibodies from Bioss: goat anti-rabbit IgG antibody (H + L), FITC-conjugated (bs-0295G-FITC); goat anti-rat IgG antibody (H + L), cyanine 3-conjugated (bs-0293G-Cy3); antibodies from Biolegend: APC anti-human CD34 antibody (#343509), FITC anti-human CD146 antibody (#361011).

Techniques: Expressing, DNA Methylation Assay, Activation Assay, Phospho-proteomics, Biomarker Discovery