Fig. 3: Keap1 regulates non-homologous end joining. | Cell Death & Disease

Fig. 3: Keap1 regulates non-homologous end joining.

From: Keap1 inhibition sensitizes head and neck squamous cell carcinoma cells to ionizing radiation via impaired non-homologous end joining and induced autophagy

Fig. 3

A Effect of Keap1 inhibition on residual γH2AX (light gray) and DNA-PKcs S2056 (dark gray) foci in HNSCC cell lines after sham or 2-Gy irradiation. B Representative immunofluorescence images of residual γH2AX (red) and DNA-PKcs S2056 (green) after indicated treatments (bar, 10 µm). C GFP-based reporter assays for homologous recombination (HR) and non-homologous end joining (NHEJ). Cal33 cells stably transfected with DRGFP or pimEJ5GFP recombinant plasmids were treated with ML334 (EC50) or DMSO. The number of GFP-positive cells was analyzed by flow cytometry. D Cal33 cells stably transfected with DRGFP or pimEJ5GFP recombinant plasmids were depleted of Keap1 by siRNA-mediated knockdown. The number of GFP-positive cells was analyzed by FACS. E Cal33 cells stably transfected with pimEJ5GFP recombinant plasmids were treated with EC10, EC25, EC50 and EC75 of ML334 (DMSO used as control). The number of GFP-positive cells was analyzed by FACS. F Cal33 cells stably transfected with pimEJ5GFP recombinant plasmids were depleted of Keap1 and NRF2 by knockdown. Results are compared to ML344 (EC50). The number of GFP-positive cells was analyzed by flow cytometry. G Representative dot plot figures of cell cycle distribution of UTSCC14 cells upon Keap1 inhibition. H Flow cytometry-based quantification of cell cycle distribution at 6 and 24 h post 6 Gy X-ray irradiation and ML334. Data are presented as mean ± SD (n = 3; two-sided t-test; **P < 0.01, ***P < 0.001; n.s., not significant (P ≥ 0.05)).

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