Fig. 3

Hypoxia probe (LOX-1), HIF-1α expression, localization, cell cycle, and Ki67-expression analyses of COL-XFM and XFM cultures. a LOX-1 phosphorescence and HIF-1α immunohistostaining of the multilayered COL-XFM and XFM cultures on D20. All nuclei are shown in magenta (DAPI). Scale bars, 100 μm in the panels of LOX-1 and 50 μm in the panels labeled “Merge.” b Immunocytostaining of COL-XFM and XFM cells treated with cobalt chloride to induce hypoxia; single-cell analysis of HIF-1α localization. The single channels (HIF-1α, DAPI, and vimentin) are shown in gray. The fluorescence intensities of HIF-1α (green) and nuclei (DAPI, magenta) are indicated by the dashed line (yellow) depicted in a single cell; cell morphology was visualized using vimentin staining (red on merged images). Scale bars, 50 μm. c Cell cycle analysis using flow cytometry in COL-XFM and XFM cells on days 10 (D10; confluence), 14, and 20 (D14 and D20; overconfluence) post-seeding. Representative histograms are shown. The percentage of the cells in G0/G1, S, and G2/M phases of cell cycle is quantified and presented in the panel on the right. d Western blot and quantification of Ki67 expression in COL-XFM and XFM cells on D10, D14, and D20. β-actin was used as an internal control to normalize protein expression of Ki67. *p < 0.05; **p < 0.01