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Primary B-CLL cells (A, B) and B-cell cancer lines (C, D) treated with GSK-7975A (GSK, 10, 20, 50 μM) for 48 hours. Alive and dead cells were determined using LIVE/DEAD® staining Kits and analyzed by flow cytometry. (A, C) Representative flow cytometry plots. (B, D) Quantification of the percentage of dead cells in (A, C). Cell mortality was also determined by flow cytometry after annexin V/PI staining (E, F). The percentage of total apoptotic cells was calculated as the percentage of annexin V-FITC positive plus annexin V-FITC/PI-positive population. (G, <t>H)</t> <t>Caspase-3</t> activity determined using <t>Ac-DEVD-AFC</t> as substrate after 24 hours with GSK-7975A (GSK, 0-50 μM) or Synta66 (S66, 0-50 μM) in primary B-CLL cells and B-cell cancer lines. Data from B-cell cancer lines are representative of at least 3 independent experiments. Statistical significance was analyzed by two-tailed, unpaired Student’s t test.
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Primary B-CLL cells (A, B) and B-cell cancer lines (C, D) treated with GSK-7975A (GSK, 10, 20, 50 μM) for 48 hours. Alive and dead cells were determined using LIVE/DEAD® staining Kits and analyzed by flow cytometry. (A, C) Representative flow cytometry plots. (B, D) Quantification of the percentage of dead cells in (A, C). Cell mortality was also determined by flow cytometry after annexin V/PI staining (E, F). The percentage of total apoptotic cells was calculated as the percentage of annexin V-FITC positive plus annexin V-FITC/PI-positive population. (G, H) Caspase-3 activity determined using Ac-DEVD-AFC as substrate after 24 hours with GSK-7975A (GSK, 0-50 μM) or Synta66 (S66, 0-50 μM) in primary B-CLL cells and B-cell cancer lines. Data from B-cell cancer lines are representative of at least 3 independent experiments. Statistical significance was analyzed by two-tailed, unpaired Student’s t test.

Journal: bioRxiv

Article Title: Blocking Orai1 constitutive activity inhibits B-cell cancer migration and synergistically acts with drugs to reduce B-CLL cell survival

doi: 10.1101/2023.12.12.571243

Figure Lengend Snippet: Primary B-CLL cells (A, B) and B-cell cancer lines (C, D) treated with GSK-7975A (GSK, 10, 20, 50 μM) for 48 hours. Alive and dead cells were determined using LIVE/DEAD® staining Kits and analyzed by flow cytometry. (A, C) Representative flow cytometry plots. (B, D) Quantification of the percentage of dead cells in (A, C). Cell mortality was also determined by flow cytometry after annexin V/PI staining (E, F). The percentage of total apoptotic cells was calculated as the percentage of annexin V-FITC positive plus annexin V-FITC/PI-positive population. (G, H) Caspase-3 activity determined using Ac-DEVD-AFC as substrate after 24 hours with GSK-7975A (GSK, 0-50 μM) or Synta66 (S66, 0-50 μM) in primary B-CLL cells and B-cell cancer lines. Data from B-cell cancer lines are representative of at least 3 independent experiments. Statistical significance was analyzed by two-tailed, unpaired Student’s t test.

Article Snippet: As reported and described previously [ ], activity of caspase-3 was performed by using Ac-DEVD-AFC substrate (Enzo Life Sciences, France).

Techniques: Staining, Flow Cytometry, Activity Assay, Two Tailed Test