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vx 970 m6620  (MedChemExpress)
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MedChemExpress vx 970 m6620
Replication catastrophe leads to increased A3B. ( A ) RKO cells were exposed to the indicated oxygen treatments for 20 h or HU (2 mM, 20 h) and co-stained for RPA, A3B and DAPI. The cells were visualized by immunofluorescence and representative images are shown. ( B ) Quantification of average A3B nuclear intensity and average number of RPA foci per cell. Each dot represents a field of view containing at least 20 cells, a minimum of 200 cells were quantified per condition. ( C ) RKO cells were pretreated with DMSO or roscovitine (Ros) for 30 min (20 μM) and exposed to 21% O 2 , cyclic hypoxia, or HU (2 mM) for 20 h. Cells were stained by immunofluorescence to detect cells with >5 53BP1 and >6 RPA foci and mounted in DAPI. ( D ) Representative images of data shown in C. ( E ) Cells were treated as in C. and western blotting was carried out. ( F ) RKO cells were pre-treated <t>with</t> <t>VX-970</t> (1 μM), AZD6738 (5 μM), or DMSO for 30 min and then exposed to 20 h cyclic hypoxia or HU alone (2 mM, 20 h). Western blotting was carried out. ( G ) In vitro deamination assay on RKO cells pre-treated with VX-970 (1 μM), AZD6738 (5 μM) or DMSO for 30 min and then exposed to 20 h cyclic hypoxia or HU alone (2 mM, 20 h). S = substrate band, P = product band. ( H ) Quantification of the bottom band (P) intensity in samples from part G. ( I ) A schematic showing how cells are treated in part J. ( J ) RKO cells were pre-treated with VX-970 for 30 min (1 μM) or DMSO and then exposed to cyclic hypoxia (8 or 20 h) Cells were labeled with BrdU (20 μM) 1 h prior to collection and analyzed by FACS. Quantification of the percentages of cells in each cycle phase. Data from three separate experiments ( n = 3) are displayed ± standard error of the mean (SEM) unless specified otherwise. ** P < 0.01, *** P < 0.001.
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Replication catastrophe leads to increased A3B. ( A ) RKO cells were exposed to the indicated oxygen treatments for 20 h or HU (2 mM, 20 h) and co-stained for RPA, A3B and DAPI. The cells were visualized by immunofluorescence and representative images are shown. ( B ) Quantification of average A3B nuclear intensity and average number of RPA foci per cell. Each dot represents a field of view containing at least 20 cells, a minimum of 200 cells were quantified per condition. ( C ) RKO cells were pretreated with DMSO or roscovitine (Ros) for 30 min (20 μM) and exposed to 21% O 2 , cyclic hypoxia, or HU (2 mM) for 20 h. Cells were stained by immunofluorescence to detect cells with >5 53BP1 and >6 RPA foci and mounted in DAPI. ( D ) Representative images of data shown in C. ( E ) Cells were treated as in C. and western blotting was carried out. ( F ) RKO cells were pre-treated with VX-970 (1 μM), AZD6738 (5 μM), or DMSO for 30 min and then exposed to 20 h cyclic hypoxia or HU alone (2 mM, 20 h). Western blotting was carried out. ( G ) In vitro deamination assay on RKO cells pre-treated with VX-970 (1 μM), AZD6738 (5 μM) or DMSO for 30 min and then exposed to 20 h cyclic hypoxia or HU alone (2 mM, 20 h). S = substrate band, P = product band. ( H ) Quantification of the bottom band (P) intensity in samples from part G. ( I ) A schematic showing how cells are treated in part J. ( J ) RKO cells were pre-treated with VX-970 for 30 min (1 μM) or DMSO and then exposed to cyclic hypoxia (8 or 20 h) Cells were labeled with BrdU (20 μM) 1 h prior to collection and analyzed by FACS. Quantification of the percentages of cells in each cycle phase. Data from three separate experiments ( n = 3) are displayed ± standard error of the mean (SEM) unless specified otherwise. ** P < 0.01, *** P < 0.001.

Journal: Nucleic Acids Research

Article Title: Replication catastrophe induced by cyclic hypoxia leads to increased APOBEC3B activity

doi: 10.1093/nar/gkab551

Figure Lengend Snippet: Replication catastrophe leads to increased A3B. ( A ) RKO cells were exposed to the indicated oxygen treatments for 20 h or HU (2 mM, 20 h) and co-stained for RPA, A3B and DAPI. The cells were visualized by immunofluorescence and representative images are shown. ( B ) Quantification of average A3B nuclear intensity and average number of RPA foci per cell. Each dot represents a field of view containing at least 20 cells, a minimum of 200 cells were quantified per condition. ( C ) RKO cells were pretreated with DMSO or roscovitine (Ros) for 30 min (20 μM) and exposed to 21% O 2 , cyclic hypoxia, or HU (2 mM) for 20 h. Cells were stained by immunofluorescence to detect cells with >5 53BP1 and >6 RPA foci and mounted in DAPI. ( D ) Representative images of data shown in C. ( E ) Cells were treated as in C. and western blotting was carried out. ( F ) RKO cells were pre-treated with VX-970 (1 μM), AZD6738 (5 μM), or DMSO for 30 min and then exposed to 20 h cyclic hypoxia or HU alone (2 mM, 20 h). Western blotting was carried out. ( G ) In vitro deamination assay on RKO cells pre-treated with VX-970 (1 μM), AZD6738 (5 μM) or DMSO for 30 min and then exposed to 20 h cyclic hypoxia or HU alone (2 mM, 20 h). S = substrate band, P = product band. ( H ) Quantification of the bottom band (P) intensity in samples from part G. ( I ) A schematic showing how cells are treated in part J. ( J ) RKO cells were pre-treated with VX-970 for 30 min (1 μM) or DMSO and then exposed to cyclic hypoxia (8 or 20 h) Cells were labeled with BrdU (20 μM) 1 h prior to collection and analyzed by FACS. Quantification of the percentages of cells in each cycle phase. Data from three separate experiments ( n = 3) are displayed ± standard error of the mean (SEM) unless specified otherwise. ** P < 0.01, *** P < 0.001.

Article Snippet: Inhibitors/drugs used were: VX-970 (M6620) (MedChemExpress), AZD6738 (MedChemExpress), Gö6976 (Sigma Aldrich), MK-8776 (Selleckchem), Bay11-7085 (Tocris Biosciences), PHA-767491 (Selleckchem), N ’ acetylcysteine (NAC) (Sigma Aldrich), and roscovitine (Ros) (Selleckchem).

Techniques: Staining, Immunofluorescence, Western Blot, In Vitro, Labeling