Fig. 6. Cells containing apical Na⁺/K⁺-ATPase were defective in maintaining sodium homeostasis. (A) Polarized Hep G2 cells were loaded with non-ratio sodium indicator, Sodium Green and then treated with mock solution (CTL), tunicamycin (TM) for 12 hours, ouabain for 10 minutes, or both. The changes in fluorescence intensity before, during and after washout of the high Na⁺ solution (Bar, 10 seconds) were monitored under a fluorescence microscope, and plotted as a function of time. The mean (line) ± s.d. (dashed line) curves are shown. (B) The same experiment as in A, but the cells were loaded with the ratio sodium indicator, SBFI. Ratio imaging by the intensity of emission fluorescence excited by 340 nm and 380 nm was plotted as a function of time. The pharmacological and mutagenesis treatments are as indicated. (C) The quantification of intracellular sodium concentrations for the data set shown in B, in vivo calibration for SBFI loading was performed. Cells treated with tunicamycin and ouabain (^*), or transfected with three deglycosylated ß-subunit mutant constructs (+), all exhibited a higher [Na⁺]_i than the control cells before the high-Na⁺ challenge (P<0.05 by Student's t-test). Means ± s.d. are shown.