Journal: Frontiers in Oncology

Article Title: Chloroquine-induced DNA damage synergizes with DNA repair inhibitors causing cancer cell death

doi: 10.3389/fonc.2024.1390518

Figure Lengend Snippet: Synergistic effect of chloroquine and KU-57788 in in various cancer cell lines. Cells were treated for 72 h with the indicated concentrations of CQ and KU-57788 at a constant ratio and survival was assessed by flow cytometry after staining with Annexin-/PI-. CI values less than 1 indicated a synergistic effect. These values were calculated using Compusyn Software.

Article Snippet: KU-57788 (KU), NU-7026 (NU) and Mirin were obtained from MedChemExpress (South Brunswick Township, NJ, USA), and Panobinostat (LBH) was provided by Novartis Pharmaceuticals, Basel, Switzerland.

Techniques: Flow Cytometry, Staining, Software

Journal: The Journal of cell biology

Article Title: A new method for high-resolution imaging of Ku foci to decipher mechanisms of DNA double-strand break repair.

doi: 10.1083/jcb.201303073

Figure Lengend Snippet: Figure 5. CSK+R extraction allows assessment of DSB repair through software-assisted Ku focus quantification. (A) Graph representing a dose–response analysis of the number of Ku foci 5 min after the indicated x-ray doses. U2OS cells were treated, postincubated for 5 min, preextracted with CSK+R, and processed. High-resolution pictures of >20 cells were acquired for each condition and submitted to automated focus detection by Volocity software. The slope given by linear regression of these data (R2 = 0.962) equated to 24 Ku foci per Gy, which is less than the expected 30–40 DSBs per Gy per mam- malian cell (Ciccia and Elledge, 2010). This apparent discrepancy might reflect some DSBs being blocked and unable to recruit Ku within the time frame of our experiments. Such blocks could represent certain DSBs possessing secondary structures or DNA base adducts, which must be removed for Ku to bind, and/or those residing within chromatin structures, which must be remodeled before Ku and other NHEJ components can gain access to the associated DNA ends. (B) Kinetic analysis of Ku IRIF numbers after 10 Gy of IR. U2OS cells were untreated (NT) or treated with 10 Gy of IR and postincubated for the indicated times. Cells were then processed and analyzed as in A. (C) Kinetic analysis of Ku IRIF numbers after 10 Gy of IR. U2OS cells were preincubated with DMSO, NU7441 (DNA-PKi), and/or KU55933 (ATMi) and then untreated (NT) or treated and analyzed as in B. (D) Impact of ATMi and/or DNA-PKi on survival after IR. U2OS cells were preincubated with DMSO, DNA-PKi, and/or ATMi and treated with the indicated IR doses. Inhibitors were washed away 18 h after treatment, and cell survival was determined by colony formation. For all graphs, each point corresponds to at least three independent experiments, vertical bars correspond to standard deviations, and asterisks indicate a significant difference to DMSO control (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Article Snippet: ATMi (KU55933) and DNA-PKi (NU7441), both obtained from Tocris Bioscience, were used at 10 and 3 μM, respectively.

Techniques: Extraction, Software, Control

Journal: Molecular Cancer

Article Title: SALL1 functions as a tumor suppressor in breast cancer by regulating cancer cell senescence and metastasis through the NuRD complex

doi: 10.1186/s12943-018-0824-y

Figure Lengend Snippet: SALL1 over-expression in breast cancer cells induces tumor cell senescence and ATM-associated DNA damage response. a and b Transfection of SALL1, but not SALL4 in MCF-7, MDA-MB-231 and E0771 breast cancer cells significantly induced the increased SA-β-Gal + cells. In contrast, over-expression of SALL1 in B16F0 melanoma cells did not increase senescent cell populations. Transfected tumor cells were cultured for an additional 5 days. Senescent cells were analyzed using the SA-β-Gal activity assay and the SA-β-Gal + tumor cells were identified with dark blue granules as indicated by the arrows (in a ). Data shown in ( b ) are mean ± SD from three independent experiments with similar results. ** p < 0.01 compared with the vector control group. c SALL1 expression in breast cancer cells induced phosphorylated activation of ATM in the transfected cells. Transfected tumor cells were determined for the p-ATM expression after culture for 3 additional days using FACS analyses. d Pretreatment of breast cancer cells with an ATM specific inhibitor KU55933 significantly prevented the induction of tumor cell senescence induced by SALL1 expression. Tumor cells were pretreated with or without KU55933 (20 μM) for 1 day, and then transfected with SALL1. SA-β-Gal expression in the transfected tumor cells was determined with SA-β-Gal staining after culture for 3 additional days. Data shown are mean ± SD from three independent experiments, and paired t-test was performed. ** p < 0.01, compared with the medium only control group. e Knockdown of ATM gene by shRNAs in MCF-7, MDA-MB-231 and E0771 cells dramatically blocked SALL1-induced tumor cell senescence. Breast cancer cells were transfected with lenti-shRNAs specific for ATM or control shRNAs. Transduced cancer cells were then transfected with SALL1 and cultured for 5 days. The SA-β-Gal + cancer cells were determined. ** p < 0.01, compared with the group transduced with the control shRNA. Data shown are representative of three independent experiments with similar results

Article Snippet: For some experiments, SA-β-Gal + populations were determined in the transfected tumor cells after exposure to various inhibitors or combined transfection with shRNAs: ATM inhibitor KU55933 (20 μM, Tocris Bioscience); mTOR inhibitor Rapmycin (5 μM, Sigma); MAPK inhibitors U0126 (10 μM), SB203580 (10 μM) and SP600125 (10 μM), or PI3 Kinase inhibitor Wortminnin (10 μM) (Calbiochemistry), or transfection with shRNAs against p38, ERK and mTOR, for 3 or 5 days.

Techniques: Over Expression, Transfection, Cell Culture, Activity Assay, Plasmid Preparation, Control, Expressing, Activation Assay, Staining, Knockdown, Transduction, shRNA

Journal: The Journal of Cell Biology

Article Title: DNA damage promotes microtubule dynamics through a DNA-PK-AKT axis for enhanced repair

doi: 10.1083/jcb.201911025

Figure Lengend Snippet: c-NHEJ pathway is indispensable for DMSR. (A) DMSR was examined when cells were treated with DNA-PK, ATM, or ATR inhibitors. Quantitation of microtubule length is shown ( n > 50). (B) DMSR was examined in cells with depletion of indicated genes. Quantitation of microtubule length is shown ( n > 50). Cells were transfected with an equal amount of scrambled siRNA as an NC. (C) 53BP1 regulated DMSR. DMSR was examined in cells with depletion of indicated genes. Quantitation of microtubule length is shown ( n > 50). (D) Depletion of Ligase 4, XRCC4, or XLF increases the extent of DMSR. Quantitation of microtubule length is shown ( n > 50). (E) Depletion of Ligase 4 prolongs the time course of DMSR. Quantitation of microtubule length is shown ( n > 50). (F) Box plot shows quantitative analysis of GFP-EB3 comet velocity in live siRNA-treated GFP-EB3–expressing G1 RPE-1 cells ( n > 30). (G) Centrosome-dependent microtubule nucleation capacity was determined by relative centrosomal GFP-EB3 intensity in live siRNA-treated GFP-EB3–expressing G1 RPE-1 cells, and quantitative analysis is shown in box plot ( n > 30). ****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05; ns, not significant. A.U., arbitrary units; MT, microtubule; UT, untreated.

Article Snippet: The following chemicals were used: DNA-PKcs inhibitor (MCE; ku57788; 1 μM), ATM inhibitor (MCE; ku55933; 10 μM), ATR inhibitor (MCE; ADZ6783; 1 μM), AKT inhibitor (TargetMol; MK-2206; 5 μM), CHK1 inhibitor (MCE; SB 218078; 10 μM), CHK2 inhibitor (MCE; CCT241533 hydrochloride; 5 μM), PLK1 inhibitor (MCE; BI2536; 100 nM), p38/MAPK inhibitor (MCE; SB 203580; 10 μM), PDK1 inhibitor (MCE; BX-795, 10 μm; BX-912, 10 μM), bleomycin (MCE; 5 μg/ml), mitomycin C (MCE; MMC, 5 μM), camptothecin (Sigma; CPT, 1 μM), cisplatin (Sigma; 2.5 μM), and etoposide (Sigma; 1 μM).

Techniques: Quantitation Assay, Transfection, Expressing

Journal: The Journal of Cell Biology

Article Title: DNA damage promotes microtubule dynamics through a DNA-PK-AKT axis for enhanced repair

doi: 10.1083/jcb.201911025

Figure Lengend Snippet: c-NHEJ is required for DMSR. (A) The efficiency of ATR inhibitor or ATM inhibitor was confirmed by probing with pS345-CHK1 antibody or pS1981-ATM antibody, respectively. (B) The time course of DNA-PK activation after RPE-1 cells were exposed to 5 Gy IR in G1/S. DNA-PK activation was determined by immunoblotting with anti-pS2056 DNA-PKcs antibody. (C) Left: Quantitative RT-PCR shows the knockdown efficiency of indicated genes. Right: Western blot analysis shows the efficiency of indicated siRNAs. RPE-1 cells were transfected with indicated siRNAs. Cells were then collected and extracted after 48 h of transfections. The efficiency was measured by indicated antibodies. (D) Depletion of CtIP or other proteins responsible for end resection, such as EXD2, EXO1, and NBS1, at G1 phase does not affect DMSR. U2OS cells were transfected with indicated siRNAs and synchronized at G1/S phase. Cells were then exposed to 2 Gy IR and recovered for 4 h. Left: Western blot analysis shows the efficiency of indicated siRNAs. Right: Microtubule nucleation ability was determined by microtubule regrowth assay. Quantitation of microtubule length was assayed as in . This experiment and the experiment in belong to a same group of experiments. Thus, the same data for the control group was included in this figure and . (E) DMSR is significantly inhibited by depletion of 53BP1 or Ku70. U2OS cells were transfected with indicated siRNA and synchronized at G1/S phase. Cells then were exposed to 2 Gy IR and recovered for 4 h. Microtubules were stained with anti–β-tubulin antibody. Scale bar, 20 µm. (F) Western blot analysis shows the efficiency of indicated siRNAs. (G) Depletion of Ligase 4, XRCC4, or XLF, which are required for c-NHEJ, promote DMSR in U2OS cells. Microtubules (green) were stained with anti–β-tubulin antibody. Scale bar, 20 µm. (H) Western blot analysis shows the efficiency of indicated siRNAs. (I) The activation of DDR and kinetics of DNA damage repair in IR-treated U2OS control cells or siLig4 cells was determined by γH2AX foci formation ( n > 100). ****, P < 0.0001; MT, microtubule; UT, untreated.

Article Snippet: The following chemicals were used: DNA-PKcs inhibitor (MCE; ku57788; 1 μM), ATM inhibitor (MCE; ku55933; 10 μM), ATR inhibitor (MCE; ADZ6783; 1 μM), AKT inhibitor (TargetMol; MK-2206; 5 μM), CHK1 inhibitor (MCE; SB 218078; 10 μM), CHK2 inhibitor (MCE; CCT241533 hydrochloride; 5 μM), PLK1 inhibitor (MCE; BI2536; 100 nM), p38/MAPK inhibitor (MCE; SB 203580; 10 μM), PDK1 inhibitor (MCE; BX-795, 10 μm; BX-912, 10 μM), bleomycin (MCE; 5 μg/ml), mitomycin C (MCE; MMC, 5 μM), camptothecin (Sigma; CPT, 1 μM), cisplatin (Sigma; 2.5 μM), and etoposide (Sigma; 1 μM).

Techniques: Activation Assay, Western Blot, Quantitative RT-PCR, Knockdown, Transfection, Regrowth Assay, Quantitation Assay, Control, Staining