Fig. 5

Radioresistance induced by hypoxia in 3D spheroid cultures. A Spheroid imaging with LOX-1. Spheroids were cultured for 48 h, stained by the hypoxia probe LOX-1, and then imaged using a fluorescence microscope. Scale bars: 200 μm. B Immunoblot of HIF1α. Cancer cells and spheroids were cultured for 48 h after seeding, and the cytoplasmic and nuclear fractions were subjected to SDS-PAGE. GAPDH and TBP were used as controls. C The illustration to describe confocal Z-stack images of 3D spheroids. D Representative confocal fluorescence microscopy images of γH2AX foci at 30 min after irradiation of HeLa spheroids with 6 Gy of X-rays or 3 Gy of C-ion beams; γH2AX (red) and nuclear DNA stained with Hoechst 33342 (blue). Scale bars: 100 μm. Fluorescence intensities were quantified against DNA content. E Fluorescence intensity profiles of γH2AX/Hoechst 33342 and normalized LOX-1/Hoechst 33342 are shown for HeLa spheroids irradiated with 6 Gy of X-rays or 3 Gy of C-ion beams. LOX-1/Hoechst 33342 fluorescence intensities were normalized to the value of Z = 0