ib anti cdc25c pt48 rabbit cell signaling  (Cell Signaling Technology Inc)

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

Journal: Materials Today Bio

Article Title: Macrophage membrane coating enhances the therapeutic effects of Houttuynia cordata exosome-like nanovesicles against triple-negative breast cancer

doi: 10.1016/j.mtbio.2025.102604

Figure Lengend Snippet: MCELNs and CELNs induced DNA damage and cell cycle arrest in TNBC cells. (A)–(B) γ-H2AX fluorescence quantification. Scale bar = 10 μm. (C)–(D) Flow cytometry detection of cell cycle of MDA-MB-231 cells treated with MCELNs and CELNs for 24 h. (E)–(J) Fluorescence quantification of P-ATR, P-CHK1, CDC25C, CDK4, and P21. Scale bar = 100 μm. (K)–(R) The protein expression levels of γ-H2AX, p21, CDK1, CDC25C, P-ATR, and P-CHK1. DHTS and CPT served as positive controls to induce cell cycle arrest and DNA damage, respectively. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: The main materials used in this study included P-CHK1, P-ATR, CDK4, CDC25C, P21, γ-H2AX (Abclone), cleaved PARP1, full length PARP1, cleaved caspase 3, BSA (BioFroxx), BCA Protein Concentration Assay Kit (Biyotime Shanghai), SDS-PAGE protein loading buffer (BOSTER, Wuhan), Three-color prestained protein standards (Abclone), Cell Cycle Assay Kit (RedFluorescence) (Biyotime Shanghai), enhanced RIPA lysate (BOSTER, Wuhan), RPMI 1640 medium(Gibco, USA), PKH67 (MedChemExpress, USA), PEG8000 (Chron Chemicals), 5-Fluorouracil (MedChemExpress, USA), and Camptothecin (MedChemExpress, USA).

Techniques: Fluorescence, Flow Cytometry, Expressing

Journal: Materials Today Bio

Article Title: Macrophage membrane coating enhances the therapeutic effects of Houttuynia cordata exosome-like nanovesicles against triple-negative breast cancer

doi: 10.1016/j.mtbio.2025.102604

Figure Lengend Snippet: MPA counteracted MCELNs-mediated DNA damage and cell cycle arrest in TNBC cells. (A) cell viability detection by CCK8. (B)–(C) Flow cytometry determination of cell cycle of MDA-MB-231 cells treated with MCELNs for 24 h. (D)–(E) γ-H2AX expression was measured using a fluorescence microscope. Scale bar = 10 μm. (F)–(K) The expression of p-CHK1, p-ATR, CDC25C, CDK4, and P21 was detected using a fluorescence microscope. Scale bar = 100 μm. (L)–(Q) The protein expression levels of γ-H2AX, p-ATR, p-CHK1, CDC25C, p21, and CDK4 by western blot analysis. ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: The main materials used in this study included P-CHK1, P-ATR, CDK4, CDC25C, P21, γ-H2AX (Abclone), cleaved PARP1, full length PARP1, cleaved caspase 3, BSA (BioFroxx), BCA Protein Concentration Assay Kit (Biyotime Shanghai), SDS-PAGE protein loading buffer (BOSTER, Wuhan), Three-color prestained protein standards (Abclone), Cell Cycle Assay Kit (RedFluorescence) (Biyotime Shanghai), enhanced RIPA lysate (BOSTER, Wuhan), RPMI 1640 medium(Gibco, USA), PKH67 (MedChemExpress, USA), PEG8000 (Chron Chemicals), 5-Fluorouracil (MedChemExpress, USA), and Camptothecin (MedChemExpress, USA).

Techniques: Flow Cytometry, Expressing, Fluorescence, Microscopy, Western Blot

Journal: Frontiers in Pharmacology

Article Title: Eupalinolide A inhibits cancer progression and induces ferroptosis and apoptosis by targeting the AMPK/mTOR/SCD1 signalling in non-small cell lung cancer

doi: 10.3389/fphar.2025.1649314

Figure Lengend Snippet: EA induces cell cycle arrest at G2/M phase in human non-small cell lung cancer cells. (A) Volcano plot of differentially expressed genes after EA treatment. (B) Cluster analysis of differentially expressed genes after EA treatment. (C) KEEG analysis of genes in A549 and H1299 cells. (D) Cell cycle of A549 and H1299 cells analyzed by flow cytometry. (E) Percentage of A549 and H1299 cells in different phases n = 3. (F) Representative Western blots of cell cycle-related proteins, CDC25C and Cyclin B1. (G) Densitometry of Western blots in panel n = 6. * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with the Control group.

Article Snippet: The membrane was incubated with the following primary antibodies at 4 °C for 12 h: CDC25C (Proteintech; 1:1,000; cat. no. 66912-1), cyclin B1 (Proteintech; 1:1,000; cat. no. 67686-1), BAX (Proteintech; 1:1,000; cat. no. 60267), SCD-1 (Proteintech; 1:1,000; cat. no. 28678), GPX4 (Abcam; 1:1,000; cat. no. ab125066), HO-1 (Abcam; 1:1,000; cat. no. ab13248), SLC7A11 (CST; 1:1,000; cat. no. 12691S), PTGS2/COX2 (Abways; 1:1,000; cat. no. CY8852), BCL2 (Abways; 1:1,000; cat. no. CY5032), p-AMPK (Abclonal; 1:1,000; cat. no. APO871), AMPK (Abclonal; 1:1,000; cat. no. A1229), p-mTOR (CST; 1:1,000; cat. no. 5536T), mTOR (Immunoway; 1:1,000; cat. no. YT2913), and β-actin (Zhongshan Goldenbridge Bio; 1:1,000; cat. no. TA-09).

Techniques: Flow Cytometry, Western Blot, Control