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Fig. 5 | Journal for ImmunoTherapy of Cancer

Fig. 5

From: Inhibition of WEE1 kinase and cell cycle checkpoint activation sensitizes head and neck cancers to natural killer cell therapies

Fig. 5

WEE1 kinase inhibition reverses granzyme B-induced MOC2 G2/M accumulation resulting in DNA damage, DNA fragmentation and cell death. a MOC2 cells were plated in AZD1775 250 (nM) or DMSO and cell index after exposure to KIL at a 3.125:1 E:T ratio was assessed by impedance analysis. In some wells, KIL were exposed to concanamycin A (100 nM) for 4 h then washed three times before addition to MOC2 cells. % loss of cell index 24 h after the addition of KIL to MOC2 cells quantified on right. b Western blot analysis of total CDK1, p-CDK1Y15 and p-H2AXS139 after treatment of MOC2 cells as in B. Densitometry listed under each blot. Results presented are representative of three independent experiments with similar results. c MOC2 cells were exposed to AZD1775 (250 nM), streptolysin O (40 ng/mL)/Granzyme B (1.5 μg/mL) or the combination for 2 h, then analyzed for cell cycle distribution via 4D cell cycle flow analysis (DNA content, EdU uptake, p-HH3S10 staining and p-H2AXS139 staining). DMSO (volume equivalent) used for control. Representative dot plots for each treatment condition shown with histogram inserts indicating cells in mitosis (p-HH3S10 positive). d Quantification of cells within select phases of the cell cycle. e Cell cycle phase-dependent quantification of DNA damage, as measured by p-H2AXS139 staining, for each treatment condition. *, p < 0.05; **, p < 0.01; ***, p < 0.001

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