Figure 5.

Ouabain enhanced cell proliferation of HAC15 cells with the ATP1A1 L104R mutant, but did not increase aldosterone production. (a) After transduction of the ATP1A1 mutant (n = 6) or control (n = 6) vectors in HAC15 cells and incubation with or without ouabain at the indicated concentration on 96-well plates for 3 days, the amount of DNA was measured by CyQUANT Direct Cell Proliferation Assay Kit. *, p < 0.05 vs. each type of control cells. (b) After transduction of ATP1A1 mutant (n = 3) or control (n = 3) vectors in HAC15 cells and incubation with or without ouabain at the indicated concentration for 3 days, cells were serum deprived in DMEM/F12 containing 0.1% serum on 24-well plates for 24 h. Cells were incubated with fresh media with 0.1% serum for 24 h, and then aldosterone levels of the supernatant were measured. Aldosterone levels were normalized based on cellular protein levels. *, p < 0.05 vs. each control cells. (c) After transduction of ATP1A1 mutation (n = 6) or control (n = 6) and incubation with or without ouabain at indicated concentration on 96-well plates for 3 days, the amounts of DNA were measured by CyQUANT Direct Cell Proliferation Assay Kit. *, p < 0.05 vs. each control cells. (d,e) After transduction of ATP1A1 mutant (n = 3) or control (n = 3) vectors in HAC15 cells and incubation with DMEM/F12 containing 0.1% serum on 12-well plates for 24 h, cells were incubated with fresh media including 10% serum with or without 1 nM of ouabain for 5 m. *, p < 0.05 vs. control cells without ouabain. **, p < 0.05 vs. the other three type of cells.