Figure 4.

HIF-1 activity and H3K9me3 levels in a 3D spheroid model of chronic and intermittent hypoxia.A, HCT116 cells were stably transfected with a GFP reporter linked to the hypoxia response element (HRE). Cells grown as spheroids and imaged on day 0, 3, 7, and 10. B, protein expression of HIF-1α, KDM4B, KDM4C, HK2, Glut1, LDHA, and H3K9me3 in HCT116 cells grown as a monolayer or as spheroids grown over 3, 7, or 10 days. Total histone H3 is used as a loading control. C, schematic illustration of spheroid exposure to oxygen conditions using oxygen-permeable membranes and schematic illustration demonstrating how confocal microscopy is used to visualize the spheroid through multiple transverse planes. D and E, confocal images of HCT116 cells grown as spheroids over 3 days, transferred onto oxygen-permeable membranes for 24 h, and then exposed to normoxia or intermittent hypoxia over a further 18 h. D, live cell fluorescence images of spheroids expressing GFP linked to the HRE in four different transverse planes (plane 4 = membrane level). Magenta = NucRed live stain; green = GFP. E, bright field and fluorescence images of spheroids that were fixed, permeabilized, and probed with antibodies. Cyan = Histone H3 protein expression; red = H3K9me3 protein expression. Images from other transverse planes are shown in Fig. S7. F, examples of single cell analysis from individual cells attached to the membrane as a monolayer and from the periphery and core of spheroids (Zoomed in images taken from (E)). The scale bar = represents 300 μm for (A, D, and E), 50 μm for (F). G, fluorescence of H3K9me3:Histone H3 analysis from (E and F). Results are the mean ± SD of n = 20 cells. ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. Complete statistical analysis comparing fluorescence between the monolayer versus periphery versus core is presented in Table S2.