Journal: Therapeutic Advances in Gastroenterology

Article Title: The role of PARP inhibitors in BRCA mutated pancreatic cancer

doi: 10.1177/17562848211014818

Figure Lengend Snippet: Ongoing clinical trials investigating novel targeting agents.

Article Snippet: , No specific genetic targets, ARID1A/ATM loss subgroup , AZD6738 (ATR kinase inhibitor) , II , Second line or more , AstraZeneca , NCT03682289 , 19 March 2023.

Techniques: Clinical Proteomics, Mutagenesis

Journal: Nature Communications

Article Title: TDP1-independent pathways in the process and repair of TOP1-induced DNA damage

doi: 10.1038/s41467-022-31801-7

Figure Lengend Snippet: a WT and TDP1-KO cells were pre-treated with 10 µM ATRi (AZD6738), 1 µM ATMi (AZD0156), 10 µM DNA-PKi (AZD7648), or 10 µM MG132 for 1 h and then treated with 10 µM CPT for 1 h. Whole-cell extracts were prepared and subjected to Western blotting with the indicated antibodies. Experiments were repeated at least three times, and similar results were obtained. b A model of alternative DNA damage signaling and repair in TDP1-KO cells. c Flow cytometry analysis of pH2AX-S139 and DNA contents (PI staining) in WT and TDP1-KO cells that were either not treated (NT) or treated with 10 µM CPT for 1 h. Gates for pH2AX-S139-positive cells were shown. Numbers are the percentage of pH2AX-S139-positive cells (mean ± SD). Statistical data were also presented as a bar chart (mean ± SD, n = 3 biologically independent experiments). Two-tailed unpaired t test with Welch’s correction was used for statistical analysis. d as in c but pDNA-PKcs-S2056 was analyzed. Statistical data were presented as a bar chart (mean ± SD, n = 3 biologically independent experiments). Two-tailed unpaired t test with Welch’s correction was used for statistical analysis. e A flow cytometry analysis of pH2AX-S139 intensity in WT and TDP1-KO cells that were either not treated (NT) or treated with 10 µM CPT for 1 h. f Quantification of e . Mean pH2AX-S139 intensity were shown in a bar chart (mean ± SD, n = 3 biologically independent experiments). Two-tailed unpaired t test with Welch’s correction was used for statistical analysis. g as in e but pDNA-PKcs-S2056 intensity was analyzed. h Quantification of g . Mean pDNA-PKcs-S2056 intensity was shown in a bar chart (mean ± SD, n = 3 biologically independent experiments). Two-tailed unpaired t test with Welch’s correction was used for statistical analysis.

Article Snippet: The chemicals used in this study included AZD6738 (ATR kinase inhibitor, S7693; Selleck Chemicals), AZD0156 (ATM kinase inhibitor, S8375; Selleck Chemicals), KU-55933 (ATM kinase inhibitor, S1092; Selleck Chemicals), AZD7648 (DNA-PK inhibitor, S8843; Selleck Chemicals), MG132 (S2619; Selleck Chemicals), APE1 redox inhibitor E3330 (E8534-5MG; Sigma-Aldrich), APE1 inhibitor III (262017-10MG; Millipore Sigma), CPT (390238-25MG; Calbiochem), etoposide (ETO, E1383100MG; Fisher Scientific), aphidicolin (APH, A0781-5MG; Sigma-Aldrich), 5,6-dichlorobenzimidazole 1-B-D-ribofuranoside (DRB, D1916-10MG; Sigma-Aldrich), and methyl methanesulfonate (MMS, AAH5512006; Fisher Scientific).

Techniques: Western Blot, Flow Cytometry, Staining, Two Tailed Test

Journal: Development (Cambridge, England)

Article Title: DNA damage induces a kinetochore-based ATM/ATR-independent SAC arrest unique to the first meiotic division in mouse oocytes

doi: 10.1242/dev.153965

Figure Lengend Snippet: Meiotic DNA damage-induced SAC activation is independent of ATM and ATR kinases. (A) Representative γH2AX staining in the nuclei of oocytes before NEB, following addition of etoposide, or etoposide with ATMi (KU55933) and ATRi (ATR kinase inhibitor II). Scale bar: 5 µm. (B) Quantification of γH2AX levels as shown in A. Number of oocytes measured is shown in parentheses. Different letters indicate significant difference ( P <0.0001; ANOVA with Dunn's multiple comparison test). (C) Percentage of oocytes completing MI following treatment with either etoposide or DMSO vehicle before NEB. Groups were matured in the presence or absence of ATMi and ATRi, and scored for polar body extrusion. (D) Oocytes from mice that were conditional double knockouts for ATR and ATM, or floxed littermate controls, were exposed to etoposide or a vehicle control, and assessed for completion of MI. (C,D) Number of oocytes used indicated in parentheses, statistical test used was Fisher's exact (ns, not significant). Error bars indicate s.d.

Article Snippet: Other additions were nocodazole (400 nM); ZM447439 (10 µM; Bio-Techne, MN, USA), Mps1 inhibitor AZ3146 (2 µM; Bio-Techne), MG132 (10 µM), KU55933 (10 µM; Merck-Millipore, UK), bleomycin (1 µM; Abcam, UK) and ATR kinase inhibitor II (10 µM; Merck-Millipore, UK).

Techniques: Activation Assay, Staining

Journal: Oncotarget

Article Title: CGK733-induced LC3 II formation is positively associated with the expression of cyclin-dependent kinase inhibitor p21 Waf1/Cip1 through modulation of the AMPK and PERK/CHOP signaling pathways

doi:

Figure Lengend Snippet: A. PK45-p and B. PK59 cells were treated with CGK733 for 6 h before being processed for Western blot analysis using antibodies against LC3 A/B and actin. C. PK59 cells were treated with CGK733 for 6 h before being processed for immunofluorescence using an antibody against LC3 A/B. Quantification of the samples are expressed as the number of endogenous LC3 puncta per cell. D. PK45-p cells were transfected with TagGFP2-LC3 Lentivirus for 48 h and then treated with CGK733 for 6 h before observation by fluorescent microscopy. Quantification of the samples are expressed as the number of GFP-LC3 puncta per cell. Error bars represent the standard deviations from counting 30 cells in three independent experiments. ***, p < 0.001.

Article Snippet: CGK733 (sc-202964), 3-MA (sc-205596) and Compound C (sc-200689) were purchased from Santa Cruz Biotechnology.

Techniques: Western Blot, Immunofluorescence, Transfection, Microscopy

Journal: Oncotarget

Article Title: CGK733-induced LC3 II formation is positively associated with the expression of cyclin-dependent kinase inhibitor p21 Waf1/Cip1 through modulation of the AMPK and PERK/CHOP signaling pathways

doi:

Figure Lengend Snippet: A. PK59 cells were treated with CGK733 for 6 h in the presence or absence of 10 μM of CQ before analysis by immunofluorescence using an antibody against LC3 A/B. The lower figure indicates the related quantification of the endogenous LC3 puncta per cell. Error bars represent the standard deviations from counting 30 cells in three independent experiments. **, p < 0.01. B. PK45- p cells were treated with CGK733 for 6 h in the presence or absence of 10 μM of CQ before analysis by Western blot. C. PK59 cells were treated with CGK733 or rapamycin for 6 h in the presence or absence of 10 μM of CQ before analysis by Western blot. D. PK45- p cells were treated with CGK733 for 6 h before performing immunoprecipitation using an antibody against LC3 A/B. 1% of the original samples were used as inputs. E. PK45- p cells were transfected with TagGFP2-LC3 Lentivirus for 48 h and then treated with CGK733 before analysis by immunofluorescence using an antibody against p62. CQ was added in this experiment in order to easily observe autophagosomes in non-treated cells.

Article Snippet: CGK733 (sc-202964), 3-MA (sc-205596) and Compound C (sc-200689) were purchased from Santa Cruz Biotechnology.

Techniques: Immunofluorescence, Western Blot, Immunoprecipitation, Transfection

Journal: Oncotarget

Article Title: CGK733-induced LC3 II formation is positively associated with the expression of cyclin-dependent kinase inhibitor p21 Waf1/Cip1 through modulation of the AMPK and PERK/CHOP signaling pathways

doi:

Figure Lengend Snippet: A. PK45- p and PK59 cells were treated with CGK733 for 6 h before being processed for Western blot analysis. B. PK45- p cells were treated with CGK733 for 6 h after transfection with AMPK siRNA for 48 h. C. PK45- p cells were treated with CGK733 for 6 h in the presence or absence of 1 μM of PERK inhibitor (GSK2606414) before analysis by Western blot. D. PK45- p cells were treated with CGK733 for 6 h after transfection with CHOP siRNA for 48 h. E. PK45- p cells were treated with CGK733 for 6 h after transfection with AMPK and/or CHOP siRNAs for 48 h.

Article Snippet: CGK733 (sc-202964), 3-MA (sc-205596) and Compound C (sc-200689) were purchased from Santa Cruz Biotechnology.

Techniques: Western Blot, Transfection

Journal: Oncotarget

Article Title: CGK733-induced LC3 II formation is positively associated with the expression of cyclin-dependent kinase inhibitor p21 Waf1/Cip1 through modulation of the AMPK and PERK/CHOP signaling pathways

doi:

Figure Lengend Snippet: A. PK45- p cells were treated with CGK733 for 6 h after transfection with LC3 B siRNA for 48 h. B. PK45- p cells were treated with CGK733 for 6 h after transfection with EGFP-LC3 B plasmid for 48 h. C. PK45- p cells were treated with CGK733 for 6 h after the transfection with LC3 A siRNA for 48 h. D. The scheme indicates that CGK733 induced the expression of p21 Waf1/Cip1 through the LC3 B-mediated AMPK and PERK/CHOP activation.

Article Snippet: CGK733 (sc-202964), 3-MA (sc-205596) and Compound C (sc-200689) were purchased from Santa Cruz Biotechnology.

Techniques: Transfection, Plasmid Preparation, Expressing, Activation Assay