p-chk2 (c13c1) (thr-68  (Cell Signaling Technology Inc)

Journal: EMBO Reports

Article Title: Tumor acidosis-induced DNA damage response and tetraploidy enhance sensitivity to ATM and ATR inhibitors

doi: 10.1038/s44319-024-00089-7

Figure Lengend Snippet: HCT116, HT-29 and SiHa cancer cells were chronically adapted at pH 6.5 or maintained at pH 7.4 ( A , C , D ) or native HCT116 cells were acutely exposed to acidic pH e ( B ) or treated with 5-FU at the indicated doses to be compared with cancer cells adapted at pH 6.5 ( E , F ). ( A – C ) Representative immunoblots of total and phosphorylated ATM, ATR, CHK1, CHK2. Actin or tubulin was used as loading control, as indicated. ( D ) Bar graph showing the proportion of tetraploid cells. ( E , F ) Representative immunoblots of total and phosphorylated ATM, ATR, CHK1, CHK2. Actin was used as loading control, as indicated. ( G – J ) Cell viability assays in HCT116 ( G , I ) and HT-29 cancer cells ( H , J ) cultured at pH 7.4 or 6.5, and treated with the indicated doses of ATMi AZD0156 ( G , H ) or ATRi elimusertib ( I , J ) for 72 h. Data information: ( A – J ) data represent n = 3 independent biological replicates. ( D , G – J ) Bar graphs represent means ± SD with three biological replicates ( D ) or six technical replicates ( G – J ), and significance was determined using two-way ANOVA with Tukey’s multiple-comparison analysis (ns non-significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001). .

Article Snippet: Antibodies targeted against phosphorylated p-ATM (D25E5) (Ser-1981) (1:1000), ATM (D2E2) (1:1000), p-ATR (D5K8W) (Thr-1989) (1:1000), ATR (E1S3S) (1:1000), p-CHK1 (133D3) (Ser-345) (1:1000), CHK1 (2G1D5) (1:1000), p-CHK2 (C13C1) (Thr-68) (1:1000), CHK2 (1C12) (1:1000) and α/β-tubulin (1:2000) are from Cell Signaling Technologies; β-Actin (AC-15) (1:30,000) from Sigma-Aldrich.

Techniques: Western Blot, Cell Culture, Comparison

Journal: EMBO Reports

Article Title: Tumor acidosis-induced DNA damage response and tetraploidy enhance sensitivity to ATM and ATR inhibitors

doi: 10.1038/s44319-024-00089-7

Figure Lengend Snippet: Reagents and tools.

Article Snippet: Antibodies targeted against phosphorylated p-ATM (D25E5) (Ser-1981) (1:1000), ATM (D2E2) (1:1000), p-ATR (D5K8W) (Thr-1989) (1:1000), ATR (E1S3S) (1:1000), p-CHK1 (133D3) (Ser-345) (1:1000), CHK1 (2G1D5) (1:1000), p-CHK2 (C13C1) (Thr-68) (1:1000), CHK2 (1C12) (1:1000) and α/β-tubulin (1:2000) are from Cell Signaling Technologies; β-Actin (AC-15) (1:30,000) from Sigma-Aldrich.

Techniques: Membrane, Modification, Protease Inhibitor, Software, Bicinchoninic Acid Protein Assay, Viability Assay

Journal: Toxicology letters

Article Title: Mechanism study of goldenseal-associated DNA damage

doi: 10.1016/j.toxlet.2013.05.641

Figure Lengend Snippet: Effects of berberine on cell cycle of HepG2 cells. (A) Flow cytometric analysis for cell cycle distribution. Histograms shown are DNA content analyses for HepG2 cells treated with the indicated concentrations of berberine for 24 h. Treated cells were stained with propidium iodide (PI) and processed for cell cycle analysis. (B) The bar graph depicts the mean percentage of each cell cycle phase ± S.D. from four independent experiments. * p < 0.05, ** p < 0.005, and *** p < 0.001 versus the respective G1, S, or G2/M phase of DMSO-treated cells. (C) Expression of cell cycle checkpoint-related proteins. HepG2 cells were treated with the indicated concentrations of berberine for 6 h. Treated cells were lysed and subjected to Western blotting analyses with antibodies against phospho-Chk1, phosphor-Chk2, and p21 Waf1/Clp1 . β-actin was used as a loading control.

Article Snippet: Standard Western blots were performed using antibodies against total H2A.X, γ-H2A.X, p21 Waf1/Cip1 , p-ChK1 (Ser-345), p-ChK2 (Thr-68) (Cell Signaling Technology, Danvers, MA), and β-actin (internal control, Santa Cruz Biotechnology, Santa Cruz, CA) followed by a secondary antibody conjugated with horseradish peroxidase (HRP).

Techniques: Staining, Cell Cycle Assay, Expressing, Western Blot, Control