Journal: International Journal of Molecular Sciences

Article Title: BCAT1 Associates with DNA Repair Proteins KU70 and KU80 and Contributes to Regulate DNA Repair in T-Cell Acute Lymphoblastic Leukemia (T-ALL)

doi: 10.3390/ijms252413571

Figure Lengend Snippet: BCAT1–depletion induces a dysfunctional DNA damage response following etoposide treatment. ( A ) Schematic representations of the plasmids encoding full-length (WT) and truncation mutants of XRCC6 (top). vWA: von Willebrand A domain; SAP: SAF-A/B, Acinus, and PIAS domain. HEK 293T cells stably expressing epitope-tagged BCAT1 were transfected with the indicated plasmid. Cell lysates were subjected to IP with anti-FLAG beads followed by immunoblot analysis with the indicated antibodies. The arrows indicate expected positions of the respective proteins, and asterisks (*) indicate non–specific bands. ( B ) Schematic representations of the plasmids encoding full-length (WT) and truncation mutants of BCAT1 (top). N: Branched–chain amino acid aminotransferase-like N-terminal domain; AT–IV: aminotransferase class IV domain; C: Branched-chain amino acid aminotransferase-like C–terminal domain. HEK 293T cells were transfected with HA–tagged XRCC6 and the indicated BCAT1 mutant plasmids. Cell lysates were subjected to IP with anti-HA beads followed by immunoblot analysis with the indicated antibodies. The arrows indicate expected positions of the respective proteins, and asterisks (*) indicate non-specific bands. ( C – E ) CCRF–CEM T-ALL cells transduced with shCTRL or sh BCAT1 were treated with 1 µM etoposide for the indicated time. Subsequently, whole cell lysates were collected and analyzed by immunoblotting for proteins implicated in ( C , D ) the activation of the DNA damage response (pDNA-PKcs, pATM, pCHK1, pCHK2, pTP53); ( E ) DNA damage (γH2AX) and apoptosis (cleaved PARP-1). Total DNA–PKcs and ATM are shown as loading controls ( C ). Total CHK2, total TP53, and GADPH are shown as loading controls ( D , E ). Phospho-protein/protein ratios are shown (top) in each panel. A graphical representation of the phospho-protein/protein ratios is also shown for selected proteins (right panels).

Article Snippet: Antibodies against tubulin (TU-02; sc-8035), c-myc (9E10; sc-40), and p53 (DO-1; sc-126) were from Santa Cruz Biotechnology (Dallas, TX, USA); antibodies recognizing FLAG epitope (#14793), BCAT1 (#88785), KU-80 (#2180), KU-70 (#4588), phosphorylated H2AX (pS139; #9718), phosphorylated DNA-PKcs (pS2056; #68716), total DNA-PKcs (#12311), phosphorylated ATM (pS1981; #5883), total ATM (#2873), phosphorylated CHK1 (pS345; #2348), phosphorylated CHK2 (pT68; #2197), total CHK2 (#6334), phosphorylated TP53 (pS15; #12571), and GADPH (#5174) were from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Stable Transfection, Expressing, Transfection, Plasmid Preparation, Western Blot, Mutagenesis, Transduction, Activation Assay

Journal: Molecular Cancer Therapeutics

Article Title: Selective Inhibition of ATM-dependent Double-strand Break Repair and Checkpoint Control Synergistically Enhances the Efficacy of ATR Inhibitors

doi: 10.1158/1535-7163.MCT-22-0685

Figure Lengend Snippet: ATM inhibition by M3541 circumvents the M6620-induced G 1 checkpoint, resulting in aberrant cell-cycle progression and cell death. A, Quantification of ATM autophosphorylation (p-ATM Ser1981 /total ATM) in A549 cells by MSD assay after 24 hours exposure to DMSO, M3541 (1 μmol/L), M6620 (200 nmol/L), or their combination. Error bars, SEM; ****, P < 0.0001 and ***, P < 0.001 by unpaired t test. B, Western blot analysis of components of the ATM/p53 signaling pathway in A549 cells after 24-hour exposure to DMSO, M3541 (1 μmol/L), M6620 (200 nmol/L), or their combination. C, BrdU/7-AAD cell-cycle analysis in A549, A375, and H460 cells after 24 hours after treatment with DMSO, M3541 (1 μmol/L), M6620 (200 nmol/L) or their combination. D, Confluence (top row) and relative cell death (bottom row) from IncuCyte live imaging in A549 parental, p53-null, and ATM-null cells exposed to DMSO, M3541 (1 μmol/L), M6620 (200 nmol/L) or their combination. Relative cell death was calculated from the number of cells positively stained with CytoTox dye normalized to confluence. Error bars, SEM. E, Representative images from IncuCyte live imaging (10× objective) of A549 parental, p53-null, and ATM-null cells after 6-day exposure to DMSO, M3541 (1 μmol/L), M6620 (200 nmol/L), or their combination. Scale bars = 100 μmol/L. F, Bliss synergy plots generated from CellTiter-Glo assay viability results. A549 paired isogenic cell lines (parental and ATM-null) were treated for 5 days with titrations of M3541 and M6620. Bliss synergy was determined using Combenefit software.

Article Snippet: MSD MULTI-ARRAY 96-well plates (L15XA-3) were coated with capture antibodies against phosphorylated ATM (Abcam #ab208775) or total ATM (Santa Cruz Biotechnology #sc135663) and incubated at 4°C overnight.

Techniques: Inhibition, Western Blot, Cell Cycle Assay, Imaging, Staining, Generated, Glo Assay, Software

Journal: Molecular Cancer Therapeutics

Article Title: Selective Inhibition of ATM-dependent Double-strand Break Repair and Checkpoint Control Synergistically Enhances the Efficacy of ATR Inhibitors

doi: 10.1158/1535-7163.MCT-22-0685

Figure Lengend Snippet: ATM inhibition by M3541 circumvents the M6620-induced G 1 checkpoint, resulting in aberrant cell-cycle progression and cell death. A, Quantification of ATM autophosphorylation (p-ATM Ser1981 /total ATM) in A549 cells by MSD assay after 24 hours exposure to DMSO, M3541 (1 μmol/L), M6620 (200 nmol/L), or their combination. Error bars, SEM; ****, P < 0.0001 and ***, P < 0.001 by unpaired t test. B, Western blot analysis of components of the ATM/p53 signaling pathway in A549 cells after 24-hour exposure to DMSO, M3541 (1 μmol/L), M6620 (200 nmol/L), or their combination. C, BrdU/7-AAD cell-cycle analysis in A549, A375, and H460 cells after 24 hours after treatment with DMSO, M3541 (1 μmol/L), M6620 (200 nmol/L) or their combination. D, Confluence (top row) and relative cell death (bottom row) from IncuCyte live imaging in A549 parental, p53-null, and ATM-null cells exposed to DMSO, M3541 (1 μmol/L), M6620 (200 nmol/L) or their combination. Relative cell death was calculated from the number of cells positively stained with CytoTox dye normalized to confluence. Error bars, SEM. E, Representative images from IncuCyte live imaging (10× objective) of A549 parental, p53-null, and ATM-null cells after 6-day exposure to DMSO, M3541 (1 μmol/L), M6620 (200 nmol/L), or their combination. Scale bars = 100 μmol/L. F, Bliss synergy plots generated from CellTiter-Glo assay viability results. A549 paired isogenic cell lines (parental and ATM-null) were treated for 5 days with titrations of M3541 and M6620. Bliss synergy was determined using Combenefit software.

Article Snippet: Plates were incubated with equal microgram amounts of lysates for 2 hours, incubated with primary detection antibodies against total ATM (Santa Cruz Biotechnology #sc135663 or Abcam #ab199726) for 1 hour, and then incubated with secondary detection antibody (MSD #R32AC-5 for anti-mouse SULFO-Tag and MSD #R32AB-5 for anti-rabbit SULFO-Tag).

Techniques: Inhibition, Western Blot, Cell Cycle Assay, Imaging, Staining, Generated, Glo Assay, Software