Fig. 2.

Mitochondrially targeted expression of polyphosphatase (MGP) reduces levels of polyP (mouse C2C12 cells; similar results were obtained with HepG2 cells). (a) In each set of four images, three separate color channels plus their superposition are shown: green, GFP fluorescence indicating MGP expression (I); red, TMRM signal reflecting inner membrane potential (II); blue, DAPI-polyP signal (III); and superposition of the three individual channels (IV). Data are shown from an MGP-expressing cell (Left) and a nonexpressing control (Right). Below the images are the intensity profile traces (green, MGP; red, TMRM; blue, DAPI) showing fluorescence intensity along the red arrows in image IV for each cell. Note the reduced DAPI emission associated with MGP expression, reflecting a reduction in polyP. (b) Differing DAPI fluorescence emission spectra recorded from nuclear (solid line) and mitochondrial (dotted line) regions, excitation wavelength, 405 nm. These spectra resemble the distinct emissions previously reported for pure DNA and for polyP, respectively, in test-tube experiments (see Results and SI Methods). (c) Relative intensity of DAPI-polyP fluorescence (integrated, >580 nm), expressed as a percentage of the signal from nontransfected control cells (100%). Cells expressing MGP show a reduction to ≈30%, whereas the signal from cells expressing only MTS-GFP is indistinguishable from nontransfected control cells. **, P < 0.001.