RNA interference analysis of PI4P production in Huh7.5 cells.

<p>Huh7.5 cells were treated with irrelevant (mock) siRNA or siRNA targeting PI4KIIIα or PI4KIIIβ as detailed in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002576#s4" target="_blank">Materials and Methods</a>. The data were collected three days after initial siRNA transfection. (A) Confocal microscopy images of Huh7.5 cells treated with PI4KIIIα siRNA, PI4KIIIβ siRNA or mock siRNA. Cells were fixed and stained as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002576#s4" target="_blank">Materials and Methods</a>. PI4P (green) localized to the plasma membrane (PM) was detected using the plasma membrane staining protocol (upper panel) <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002576#ppat.1002576-Hammond1" target="_blank">[37]</a>. Nuclei were stained by the Hoechst dye (blue). PI4P in the intracellular membranes (IM) was revealed using the Golgi staining protocol (lower panel). Together with PI4P, Golgi membranes were stained with the Golgi marker giantin (red). Colocalization of PI4P with Golgi membranes results in yellow color. (B) Quantification of PI4P levels by immunofluorescence analysis. Changes in mean fluorescence intensity relative to the control (mock siRNA) are shown. Four randomly picked fields were analyzed per each condition, as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002576#s4" target="_blank">Materials and Methods</a>. Data are presented as averages ± SEM. *, p<0.05; **, p<0.01. (C) Immunoblot analysis of protein expression after RNAi silencing. Lysates prepared from Huh7.5 cells transfected with irrelevant siRNA (mock), PI4KIIIα siRNA (PIKA) or PI4KIIIβ siRNA (PIKB) were analyzed by immunoblotting with PI4KIIIα, PI4KIIIβ or β-actin antibodies as indicated in the figure. Positions of the protein molecular weight markers are shown on the left side.