FIG. 7.

Translocation of V-ATPase in renal epithelial cells overexpressing constitutively active PI3K. (a) LLC-PK₁ cells were incubated in standard medium containing glucose and serum on coverslips. Cells were infected with Adeno/Myr-p110-Myc or Adeno/LacZ at an MOI of 10 for 48 h. Then cells were transferred to glucose-free medium containing dialyzed serum and 0.1 mM glucose for 16 to18 h followed by stimulation with 25 mM glucose for 15 min. Then cells were fixed, permeabilized, and stained with H6.1 V-ATPase antibody. Expression of constitutively active PI3K was sufficient to maintain vesicular staining of V-ATPase in glucose-deprived cells, similarly to cells incubated in standard medium or after stimulation with glucose (see arrowheads, for example). (b) HK-2 cells were grown on coverslips in standard conditions. Infection with Adeno/Myr-p110-Myc and Adeno/LacZ, serum deprivation, and stimulation were as described above. Cells were fixed, permeabilized, and double stained with antibodies for V₁ subunit E and V_o subunit a or stained with antibody against V₁ subunit B₁. Confocal images of a and E double staining (0.5-μm optical sections) are shown together with overlays of two stainings (a/E). Overlaid images of vertical (XZ) sections for a/E double staining are also shown. (c) LLC-PK₁ cells were incubated in standard medium containing glucose and serum on coverslips. Infection with Adeno/Myr-p110-Myc and Adeno/LacZ, serum deprivation, and stimulation were as described above. LY294002 (LY) (25 μM) and vehicle were added 60 min prior to cell fixation. Then the cells were fixed, permeabilized, and stained with H6.1 V-ATPase antibody. Nuclei were counterstained with DAPI.