Journal: Cancers

Article Title: WEE1 Inhibition Enhances Anti-Apoptotic Dependency as a Result of Premature Mitotic Entry and DNA Damage

doi: 10.3390/cancers11111743

Figure Lengend Snippet: Apoptosis induced by AZD1775 in diffuse large B-cell lymphoma (DLBCL). ( A ) Cell viability analysis was measured with propidium iodide flow cytometry in DLBCL cell lines treated with AZD1775 for 72 h. Data were plotted as the mean ± SD ( n = 3). ( B ) Representative flow cytometry of apoptosis (Annexin V/Propidium iodide) induced by treatment of AZD1775 for 18 h in representative cell lines SUDHL-10 and SC-1. ( C ) Mitochondrial response of DLBCL cell lines treated with AZD1775 for 18 h, plotted as the delta mitochondrial outer membrane permeabilization (ΔMOMP%). ΔMOMP% was calculated by subtracting the percentage treated MOMP from percentage untreated MOMP. Cell line SUDHL-10 was treated with 0.25 µM AZD1775, cell lines OCI-LY3, U-2932, SUDHL-2, and SUDHL-5 were treated with 0.5 µM AZD1775 and cell lines SUDHL-4, SUDHL-6, and SC-1 were treated with 1 µM AZD1775. Data were plotted as the mean ± SD ( n = 3). Statistical analysis was performed using a one-sample t -test as compared to untreated control cells (* p ≤ 0.05).

Article Snippet: A cell suspension was mixed 1:1 with 4 µM JC-1 permeabilization/staining solution ((ENZ-52304, Enzo Life Sciences, Farmingdale, NY, USA), 0.004% digitonin (1500 643, Boehringer Mannheim, Mannheim, Germany), 20 mM β-mercaptoethanol (8.05740, Merck) and 40 µg/mL oligomycin (O4876, Sigma Aldrich) prepared in MEB) and incubated at room temperature in the dark for 10 min. BIM, PUMA, BAD, NOXA, MS1, HRK, BMF, and PUMA2A (JPT Peptide Technologies, Berlin, Germany) were prepared in MEB in a black flat-bottom non-treated polystyrene 96-well plate (3915 Costar, Corning Incorporated, Kennebunk, ME, USA).

Techniques: Flow Cytometry

Journal: Cancers

Article Title: WEE1 Inhibition Enhances Anti-Apoptotic Dependency as a Result of Premature Mitotic Entry and DNA Damage

doi: 10.3390/cancers11111743

Figure Lengend Snippet: Rescue of AZD1775-induced cellular effects by CDK inhibitors RO3306, palbociclib and roscovitine. ( A ) Representative examples of apoptotic cells, cell cycle, and γH2AX flow cytometry analysis of SC-1 treated with 10 µM RO3306, 1 µM palbociclib, 15 µM roscovitine, and 1 µM AZD1775 after 18 h of incubation. ( B ) Dynamic BH3 profile for 0.3 µM BIM, 10 µM NOXA, 10 µM HRK, and 0.1 µM BAD of SC-1 cells treated with RO3306, palbociclib, roscovitine, and AZD1775 after 18 h of incubation. Delta mitochondrial outer membrane permeabilization (ΔMOMP%) was calculated by subtracting the percentage treated MOMP from percentage untreated MOMP. Data were plotted as the mean ± SD ( n = 3). Statistical analysis was performed using a one-sample t -test as compared to untreated control cells (* p ≤ 0.05).

Article Snippet: A cell suspension was mixed 1:1 with 4 µM JC-1 permeabilization/staining solution ((ENZ-52304, Enzo Life Sciences, Farmingdale, NY, USA), 0.004% digitonin (1500 643, Boehringer Mannheim, Mannheim, Germany), 20 mM β-mercaptoethanol (8.05740, Merck) and 40 µg/mL oligomycin (O4876, Sigma Aldrich) prepared in MEB) and incubated at room temperature in the dark for 10 min. BIM, PUMA, BAD, NOXA, MS1, HRK, BMF, and PUMA2A (JPT Peptide Technologies, Berlin, Germany) were prepared in MEB in a black flat-bottom non-treated polystyrene 96-well plate (3915 Costar, Corning Incorporated, Kennebunk, ME, USA).

Techniques: Flow Cytometry, Incubation

Journal: Cancers

Article Title: WEE1 Inhibition Enhances Anti-Apoptotic Dependency as a Result of Premature Mitotic Entry and DNA Damage

doi: 10.3390/cancers11111743

Figure Lengend Snippet: Dynamic BH3 profile and validation experiments in a DLBCL patient treated with AZD1775. ( A ) Static BH3 profile and ( B ) dynamic BH3 profile of DLBCL patient cells treated with 0.5 µM AZD1775 for 18 h. Alamethicin and PUMA2A were used as positive and negative controls, respectively. Delta mitochondrial outer membrane permeabilization (ΔMOMP%) was calculated by subtracting the percentage treated MOMP from percentage untreated MOMP. ( C ) Cell viability flow cytometry data for DLBCL patient cells pre-treated with AZD1775 for 18 h and incubated with increasing concentrations of venetoclax and ( D ) navitoclax for 24 h. Data were normalized to the control.

Article Snippet: A cell suspension was mixed 1:1 with 4 µM JC-1 permeabilization/staining solution ((ENZ-52304, Enzo Life Sciences, Farmingdale, NY, USA), 0.004% digitonin (1500 643, Boehringer Mannheim, Mannheim, Germany), 20 mM β-mercaptoethanol (8.05740, Merck) and 40 µg/mL oligomycin (O4876, Sigma Aldrich) prepared in MEB) and incubated at room temperature in the dark for 10 min. BIM, PUMA, BAD, NOXA, MS1, HRK, BMF, and PUMA2A (JPT Peptide Technologies, Berlin, Germany) were prepared in MEB in a black flat-bottom non-treated polystyrene 96-well plate (3915 Costar, Corning Incorporated, Kennebunk, ME, USA).

Techniques: Flow Cytometry, Incubation