FIGURE 7.

PI(4,5)P₂ and PI(3,4,5)P₃ differentially regulate NHE1-dependent cell survival. LLC-PK1 cells (A), as well as wild-type (B, D, and E) and NHE1-null mouse proximal tubule cell lines (C, F, and G) were cultured on permeable supports. LLC-PK1 cells underwent PI(4,5)P₂ or PI(3,4,5)P₃ sequestration by overexpressing GFP-tagged PLCδ-PH or Akt-PH peptide constructs, respectively (A), or preincubation with PI 3-kinase inhibitor LY-294002, Akt inhibitor Akt VIII, or NHE1 inhibitor EIPA (B and C). Mouse PTC were loaded with PI(4,5)P₂ or PI(3,4,5)P₃ vesicles (D–G). All groups were induced to undergo apoptosis with staurosporine (STS, 1 μm, 5 h, 37 °C). Apoptosis was determined by TUNEL assay (A–D and F) or detection with antibodies that recognize the active, 17 kDa, cleaved caspase-3 by immunoblot analysis (E and G, upper panels). Blots were stripped and re-probed for GAPDH as a loading control (E and G, lower panels). *, p < 0.05 compared with STS alone by analysis of variance on ranks and the Dunnett post hoc analysis in A; *, p < 0.05 compared with all other groups by analysis of variance in B–D.