Fig. 3. MCL-1 depletion in radioresistant NPC cells inhibited stem-cell characteristic and restored radiation sensitivity.

S26 NPC cells were irradiated by daily exposure (5 days per week, for 6 weeks) to single fractions of 2 Gy X-ray radiation to acquire radioresistant cell line S26-R. A Western blotting analysis of BCL-2 family members expression in S26 and its radio-resistant S26-R cells. B–F S26-R cells stably transfected with shRNA targeting MCL1 (sh1, sh2) or scrambled shRNA (shNC) were analyzed as follows. (B, left panel) Representative images of single-cell suspensions in ultra-low-attachment culture plates are shown. (Right panel) The formed spheroids were counted via microscopy (upper) and the size of spheroids were compared (lower), **p < 0.01, Student’s t-test, Scale bar: 200 μm. C Percentages of SP cells are shown in the left panel, and the right panels compare the SP formed in S26-R cells transfected with the shRNA targeting MCL-1 or its control, n = 3; *p < 0.05, Student’s t-test. D Protein levels of stem-cell markers were determined by western blotting, tubulin was used as the loading control. E Cell viability of S26-R cells transfected with shRNA targeting MCL-1 or its control after treatment with ionizing radiation determined by the MTS assay, **p < 0.01, Student’s t-test. F Cell survival curve for treatment with increased doses of ionizing radiation in S26-R cells transfected with shRNA targeting MCL-1 or its control, *p < 0.05, **p < 0.01, Student’s t-test. G, H S26-R cells transfected with shRNA targeting MCL1 or its control were treated with indicated dose of radiation, then necrotic cells are revealed by propidium iodide staining (G) and apoptotic cells are revealed by flow cytometry analysis (H). Qualification results are shown, **p < 0.01, Student’s t-test. (NPC, nasopharyngeal carcinoma; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; shRNA, short hairpin RNA; SP, side population).