Journal: Trends in molecular medicine

Article Title: Death by releasing the breaks: CHK1 inhibitors as cancer therapeutics

doi: 10.1016/j.molmed.2010.10.009

Figure Lengend Snippet: Structure and kinase selectivity of clinically advanced CHK1 inhibitors.

Article Snippet: AZD7762 AZD7762 is a potent and selective urea-based CHK1/CHK2 inhibitor discovered at AstraZeneca (Waltham, MA, USA) by medicinal chemistry optimization of a high-throughput screening hit [ 48 ].

Techniques:

Journal: Science Advances

Article Title: Phosphorylation of human TRM9L integrates multiple stress-signaling pathways for tumor growth suppression

doi: 10.1126/sciadv.aas9184

Figure Lengend Snippet: ( A ) Simplified schematic of the MEK/ERK signaling pathways transduced through RSK. Kinase inhibitors used in this study are denoted. ( B ) H 2 O 2 -induced Thr 202 /Tyr 204 dual phosphorylation of ERK1/2 and hyperphosphorylation of TRM9L are dependent on MEK1/2 activation. HCT116 + FLAG-TRM9L cells that were untreated or exposed to 880 μM H 2 O 2 with or without the pretreatment of MEK1/2 inhibitor U0126 were harvested by mechanical dislodgement and analyzed by immunoblot for the indicated proteins. ( C and D ) H 2 O 2 -induced hyperphosphorylation of TRM9L is inhibited by pretreatment of the RSK inhibitors BI-D1870 (C) and AZD7762 (D). Top: HCT116 + FLAG-TRM9L cells were left untreated or treated with the indicated concentration of H 2 O 2 in the presence of vehicle [dimethyl sulfoxide (DMSO)] or the indicated RSK inhibitor followed by immunoblot analysis. Bottom: Quantification of H 2 O 2 -induced hyperphosphorylation of Ser 380 in the presence of DMSO or RSK inhibitor. Data represent means ± SD ( n = 3).

Article Snippet: After overnight incubation, kinase inhibitors diluted in DMSO were added: U0126 (10 μM), a selective MEK1/2 inhibitor (Cell Signaling Technology); AZD7762 (150 nM), a selective inhibitor of checkpoint kinases 1 (Chk1) and Chk2, which also inhibits RSK2/3 (Cayman Chemicals); and BI-D1870 (10 μM), a potent inhibitor of the N-terminal kinase domain of RSK 1, 2, 3, and 4 (Cayman Chemicals).

Techniques: Activation Assay, Western Blot, Concentration Assay

Journal: Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine

Article Title: Feedback regulation of methyl methanesulfonate and ultraviolet-sensitive gene clone 81 via ATM/Chk2 pathway contributes to the resistance of MCF-7 breast cancer cells to cisplatin.

doi: 10.1177/1010428317694307

Figure Lengend Snippet: Figure 3. Effects of ATM and Chk2 inhibitors on cell proliferation in response to cisplatin. MCF-7 cells were pretreated with ATM (KU55933, 10 µM) and Chk1/Chk2 (AZD7762, 300 nM) inhibitors for 16 h, then different concentrations of cisplatin (0.5, 2, 4, 8, and16 µg/mL) were added in combination with KU55933 and AZD7762 for 24 h. Cell viability was evaluated by CCK-8 assay: (a) effect of ATM inhibitors on cell proliferation under cisplatin treatment and (b) effect of Chk2 inhibitors on cell proliferation under cisplatin treatment. Statistical analysis was performed comparing treated cells with controlled cells. *p < 0.05.

Article Snippet: ATM inhibitor (KU55933, ab120637) and Chk2 inhibitor (AZD7762, S1532) were provided by Abcam (Cambridge, UK) and Selleck (Houston, TX, USA), respectively.

Techniques: CCK-8 Assay

Journal: Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine

Article Title: Feedback regulation of methyl methanesulfonate and ultraviolet-sensitive gene clone 81 via ATM/Chk2 pathway contributes to the resistance of MCF-7 breast cancer cells to cisplatin.

doi: 10.1177/1010428317694307

Figure Lengend Snippet: Figure 4. Treatment with cisplatin activates the ATM/Chk2 pathway and upregulates Mus81 expression in MCF-7 cells: (a) MCF-7 cells were exposed to different concentrations of cisplatin for 24 h. Total proteins were extracted for immunoblotting. β-actin served as a loading control, and (b) relative protein expression levels were analyzed by Quantity One analysis software. pATM-S1981: phosphorylated ATM (Ser1981); pChk2-T68: phosphorylated Chk2 (threonine 68); pP53-S15: phosphorylated P53 (Serine15); PBS: phosphate buffer saline. *p < 0.05.

Article Snippet: ATM inhibitor (KU55933, ab120637) and Chk2 inhibitor (AZD7762, S1532) were provided by Abcam (Cambridge, UK) and Selleck (Houston, TX, USA), respectively.

Techniques: Expressing, Western Blot, Control, Software, Saline

Journal: Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine

Article Title: Feedback regulation of methyl methanesulfonate and ultraviolet-sensitive gene clone 81 via ATM/Chk2 pathway contributes to the resistance of MCF-7 breast cancer cells to cisplatin.

doi: 10.1177/1010428317694307

Figure Lengend Snippet: Figure 5. Cisplatin upregulates Mus81 by activating ATM/Chk2 pathway in MCF-7 cells. MCF-7 cells were pretreated with ATM inhibitor (KU55933, 10 µM) and Chk2 inhibitor (AZD7762, 300 nM) for 16 h. A total of 1 µg/mL cisplatin was added in combination with them for 24 h: (a) total proteins were extracted for immunoblotting to detect pATM-S1981, pChk2-T68, Mus81, and β-actin. β-actin served as a loading control, and (b) total mRNA were extracted for RT-PCR to detect Mus81 mRNA levels. **p < 0.001.

Article Snippet: ATM inhibitor (KU55933, ab120637) and Chk2 inhibitor (AZD7762, S1532) were provided by Abcam (Cambridge, UK) and Selleck (Houston, TX, USA), respectively.

Techniques: Western Blot, Control, Reverse Transcription Polymerase Chain Reaction

Journal: Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine

Article Title: Feedback regulation of methyl methanesulfonate and ultraviolet-sensitive gene clone 81 via ATM/Chk2 pathway contributes to the resistance of MCF-7 breast cancer cells to cisplatin.

doi: 10.1177/1010428317694307

Figure Lengend Snippet: Figure 6. Mus81 knockdown activates ATM/Chk2 pathway and p53/Bcl-2 in MCF-7 cells under cisplatin treatment. MCF-7 cells were infected with shMus81 lentiviruses for 48 h. A total of 1 µg/mL cisplatin was added for 24 h: (a) total proteins were extracted for immunoblotting to detect pATM-S1981, pChk2-T68, pP53-S15, Bcl-2, Mus81, and β-actin. β-actin served as a loading control, and (b) relative protein expression levels were analyzed by Quantity One analysis software. *p < 0.05.

Article Snippet: ATM inhibitor (KU55933, ab120637) and Chk2 inhibitor (AZD7762, S1532) were provided by Abcam (Cambridge, UK) and Selleck (Houston, TX, USA), respectively.

Techniques: Knockdown, Infection, Western Blot, Control, Expressing, Software

Journal: Nature Communications

Article Title: Treacle controls the nucleolar response to rDNA breaks via TOPBP1 recruitment and ATR activation

doi: 10.1038/s41467-019-13981-x

Figure Lengend Snippet: a Time-lapse microscopy of U2OS cells expressing low levels of GFP-tagged Treacle, after transfection with I-Ppo1. b Timeline of the morphological changes of the nucleoli after I-Ppo1 transfection. c Morphological changes of the nucleoli after I-Ppo1 transfection in ATMi (KU-55933) and ATRi (VE-821) treated cells. Displayed are maximum intensity projections of confocal z-stacks. NPM stands for nucleophosmin d Quantification of Treacle foci number per nuceolus, nucleolar volume and nucleolar sphericity by segmentation of 3D reconstituted z-stacks. DMSO/0 min n = 262, DMSO/60 min n = 239, DMSO/120 min n = 264, ATMi/0 min n = 174, ATMi/60 min n = 166, ATMi/120 min n = 179, ATRi/0 min n = 208, ATRi/60 min n = 218, ATRi/120 min n = 201 independent cells. Bars represent means, dotted lines within the violin plots represent median and quartiles. e ATM and ATR recruitment to sites of nucleolar DSBs in response to I-Ppo1 transfection. Displayed are maximum intensity projections of confocal z-stacks. NPM stands for nucleophosmin. f Quantification of nucleolar EU incorporation after I-Ppo1 transfection in I-Ppo1 H98A/DMSO ( n = 215), DMSO ( n = 205), ATMi (KU-55933, n = 206) and ATRi (VE-821, n = 219) treated cells. g Quantification of nucleolar EU incorporation in siCtrl I-Ppo1 H98A and WT ( n = 203) and siATR I-Ppo1 H98A and WT ( n = 210) treated cells. h Quantification of nucleolar EU incorporation after I-Ppo1 H98A ( n = 229) and I-Ppo1 WT transfection in control (DMSO, n = 231), ATRi (VE-821, n = 237), CHK1i/CHK2i (AZD7762, n = 246) and CHK1i (GDC-0575, n = 255) treated cells. f – h Boxes represent the 25–75 percentile range with median and whiskers represent the 5–95 percentile range. Data points outside of this range are shown individually. All scalebars = 10 µm. Source data are provided as a Source Data file.

Article Snippet: Unless stated otherwise, the following compounds were used at the indicated final concentrations: ATM inhibitor KU-55933 (5 μM; Selleckchem), ATR inhibitor VE-821 (5 μM; Selleckchem), ATR inhibitor HY-19323 (5 μM; MedChemExpress), CHK1/CHK2 inhibitor AZD7762 (300 nM; TargetMol), CHK1 inhibitor GDC-0575 (300 nM; MedChemExpress).

Techniques: Time-lapse Microscopy, Expressing, Transfection, Control

Journal: Nature Communications

Article Title: Treacle controls the nucleolar response to rDNA breaks via TOPBP1 recruitment and ATR activation

doi: 10.1038/s41467-019-13981-x

Figure Lengend Snippet: a Timecourse of RPA2 foci formation in U2OS cells after I-Ppo1 transfection. b Timecourse of RPA2 foci formation in cells counterstained for Cyclin A. c Quantification of the experiment in b (all data points shown, red bars represent mean). d Timecourse of RPA2 foci formation in U2OS WT and NBS1ΔN cells after I-Ppo1 transfection. e Quantification of the experiment in d (all data points shown, red bars represent mean). f Timecourse of RPA2 foci formation in U2OS cells treated with ATMi (KU-55933) and ATRi (VE-821) after I-Ppo1 transfection. g Quantification of the experiment in f (all data points shown, red bars represent mean). All scalebars = 10 µm. Source data are provided as a Source Data file.

Article Snippet: Unless stated otherwise, the following compounds were used at the indicated final concentrations: ATM inhibitor KU-55933 (5 μM; Selleckchem), ATR inhibitor VE-821 (5 μM; Selleckchem), ATR inhibitor HY-19323 (5 μM; MedChemExpress), CHK1/CHK2 inhibitor AZD7762 (300 nM; TargetMol), CHK1 inhibitor GDC-0575 (300 nM; MedChemExpress).

Techniques: Transfection

Journal: Nature Communications

Article Title: Treacle controls the nucleolar response to rDNA breaks via TOPBP1 recruitment and ATR activation

doi: 10.1038/s41467-019-13981-x

Figure Lengend Snippet: a Timecourse of TOPBP1 localization in GFP-Treacle expressing U2OS cells after I-Ppo1 transfection. b TOPBP1 localization 2 h after I-Ppo1 transfection in NBS1ΔN cells and NBS1ΔN cells complemented with wild-type and mutant NBS1-mNG (percentage of cells with > 2 TOPBP1 caps are indicated; one of two experiments is shown). c TOPBP1 localization 2 h after I-Ppo1 transfection in ATMi- and ATRi-treated cells versus vehicle (DMSO)-treated cells (percentage of cells with TOPBP1 foci inside of the nucleoli are indicated; one of two experiments is shown). d Quantification of cells with TOPBP1 caps after ATMi and ATRi treatment versus vehicle (DMSO) treatment (red bars represent mean). e Quantification of nucleolar EU incorporation after I-Ppo1 H98A ( n = 692) and I-Ppo1 WT ( n = 649) transfection in control U2OS and after I-Ppo1 H98A ( n = 450) and I-Ppo1 WT ( n = 521) transfection in siTOPBP1-treated cells (boxes represent the median with 25–75 percentile range and whiskers represent the 5–95 percentile range. Data points outside of this range are shown individually). f Endogenous Treacle localization 2 h after I-Ppo1 transfection in control siRNA and TOPBP1 siRNA transfected cells (percentage of cells with > 2 Treacle caps are indicated; one of two experiments is shown). g TOPBP1 localization in Treacle-depleted U2OS cells and control cells 2 h after I-Ppo1 transfection (percentage of cells with > 2 TOPBP1 caps are indicated; one of two experiments is shown). h HA-immunoprecipitations from 293FT cells transfected with the indicated HA-tagged Treacle variants, in the presence or absence of ATMi and ATRi and with and without IR treatment as indicated. STTT mutated Treacle: S171A, T173A, T203A, T210A. All scalebars = 10 µm. Source data are provided as a Source Data file.

Article Snippet: Unless stated otherwise, the following compounds were used at the indicated final concentrations: ATM inhibitor KU-55933 (5 μM; Selleckchem), ATR inhibitor VE-821 (5 μM; Selleckchem), ATR inhibitor HY-19323 (5 μM; MedChemExpress), CHK1/CHK2 inhibitor AZD7762 (300 nM; TargetMol), CHK1 inhibitor GDC-0575 (300 nM; MedChemExpress).

Techniques: Expressing, Transfection, Mutagenesis, Control