Targeted expression of polyphosphatase (scPPX) by the NoLS of NOA36 reduces levels of nucleolar polyphosphate in HeLa cells.

<p>A) Confocal fluorescence microscopy of fixed HeLa cells transfected with a vector with: the NoLS-scPPX construct (pIRES-NoLS-scPPX), a scPPX construct (pIRES-scPPX), or an empty vector (pIRES). Specific labelling of polyphosphate (PolyP, green) and Flag antibody (NoLS-scPPX, red) was determined. A representative experiment is shown (n = 3). Bar: 10 µm. B) Densitometric analysis of the polyphosphate signal at the nucleolus in control cells (control), and in cells after the transfection procedure with the NoLS-scPPX construct (NT and NoLS-scPPX). Co-labelling with Flag antibody allows the signals of non-transfected cells (NT) and positive transfected cells (NoLS-scPPX) to be separated. Analysis was performed using ImageJ 1.43u software. Results represent the mean ± S.D. of the measurements in 150 cells in each condition (n = 3). Asterisk indicates significant differences in comparison with non transfected cells, determined by T-test (P<.002). C) Western blot expression of Flag-polyphosphatase protein in cell extracts of cells transfected with an empty vector (pIRES), or with a vector with the NoLS-scPPX construct (pIRES-NoLS-scPPX). Protein molecular weight markers are shown in the middle of the panel (MW), and their estimated protein sizes (in kDa) are indicated to the right. The expected molecular weight for the polyphosphatase protein (∼50 kDa) is indicated with an arrow. Nonspecific bands around 85 and 30 kDa appear for all analyzed samples. A representative experiment is shown (n = 3).