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

Journal: Cancers

Article Title: Precision Oncology with Drugs Targeting the Replication Stress, ATR, and Schlafen 11

doi: 10.3390/cancers13184601

Figure Lengend Snippet: Pharmacological targets and drugs inducing replication stress.

Article Snippet: Additional drugs are in early development comprising M4344 and M1774 (EMD-Serono Merck, Darmstadt, Germany), ART0380 (Artios Pharma, New York, NY, USA) and RP-3500 (Repare Therapeutics, Quebec, QC, Canada) [ ].

Techniques: Biomarker Discovery, Synthesized, Blocking Assay, Inhibition

Journal: Cancer Research

Article Title: Replication Stress Is an Actionable Genetic Vulnerability in Desmoplastic Small Round Cell Tumors

doi: 10.1158/0008-5472.CAN-23-3603

Figure Lengend Snippet: PARPi and ATRi have synergistic cytotoxic effects in models of DSRCT with high PARP1 expression. A and B, PARP1 expression ( A ) and PARylation levels ( B ) as assessed by IHC in a cohort of 16 DSRCT samples, compared with those of the JN1 and R cell lines (PARP1 and PAR expression levels are shown as H-scores). Representative cases (PARP1-high vs. PARP1-low tumors; PAR-high vs. PAR-low tumors) are shown to the right, compared with JN1 and R cells. C and D, Surface plots of Bliss independence scores calculated for the talazoparib–M4344 combination in JN1 ( C ) and R ( D ) cell lines at 7 days. E, The GR_13-PDX-O model was established from the primary peritoneal tumor of a patient with DSRCT, with confirmation of EWSR1::WT1 fusion by FISH and WT1-Cter IHC (Supplementary Fig. S8). F, Surface plot of Bliss independence scores calculated for the talazoparib–M4344 combination in the GR_13 PDX-O at 7 days. Mean ± SD; n = 3. Surface plots: the x -axis and y -axis values indicate drug concentrations, and the z -axis values indicate the associated synergy score; score < −10, antagonistic interaction; score = 0, absence of interaction; score > 10, synergistic interaction. G, Schematic illustration of an in vivo therapeutic experiment performed to evaluate the antitumor effect of PARPi talazoparib and ATRi M1774 in NSG mice engrafted with JN1 xenografts. H, Therapeutic responses to drug treatment in mice harboring JN1 xenografts. Mean tumor volume ± SD; two-way ANOVA and post hoc Dunnett test. I, Tumor volume at the time of mice sacrifice. Mean ± SD; one-way ANOVA and post hoc Šídák test. *, P < 0.01; ns, not significant. Tala, talazoparib.

Article Snippet: The ATRi tuvusertib (M1774) was provided by Merck.

Techniques: Expressing, In Vivo