Inducible inhibition of outer mitochondrial PKA antagonizes mitochondrial fusion and survival.

(A) Lysates of clonal PC12 cell lines (clone numbers listed) expressing outer-mitochondrial GFP-PKI (omPKI) or GFP-inhibitor-2 (omInh2) from a doxycycline (Dox)-inducible promoter were treated ± Dox (1 µg/ml, 48 h) or forskolin (forsk, 10 µM, 2 h) and immunoblotted for GFP, PKA substrates (RXX[pS/pT] antibody), and PP2A/C as a loading control. (B–D) PC12::omPKI and omInh2 cells were analyzed for mitochondrial morphology ± Dox induction for 2 d (representative confocal images of live cells stained with TMRM (B), reference image based length scores in (C), and digital morphometry in (D) of the same set of 31–38 cells). (E, F) PC12 cell lines were treated ± Dox for 2 d to induce omPKI or omInh2, followed by a 2 d challenge with staurosporine or H2O2. In (E), viability was scored by a colorimetric assay (tetrazolium reduction to formazan), while apoptotic nuclei were counted in (F). Bar graphs show means ± S.E.M. and are representative of at least three independent experiments. Student's t test comparisons are between ± Dox-treated cultures.