Figure 2. Kinetics of TonEBP nuclear translocation in ARPE-19 cells exposed to hyperosmolar stress. ARPE-19 cells were incubated for 4, 8, or 12 h with iso-osmolar medium (control) or media containing the additional presence of 100 mM NaCl (Na100) or 200 mM sucrose (Su200). Cells were then fixed and exposed to immunofluorescent staining of tonicity enhancer binding protein (TonEBP) (in green) as described in the Methods section. A: Negative control (CTNeg) was performed in the sole presence of secondary antibodies. B: Cells were incubated under iso-osmolar conditions for 0 h (CT). C, E, G: Cells were incubated with Na100. D, F, H: Cells were incubated with Su200. Cell nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI; blue). Scale bars represent 20 µm. Pictures were taken at 40X magnification. Data are representative of three independent experiments.