FIG 3.

The knockdown of PIP5K1α and PIP5K1γ impairs HIV-1 Pr55^Gag localization to the plasma membrane. TZM-bl HeLa cells, transfected with either a nontargeting control siRNA or with siRNAs targeting each PIP5K1 isoform, were then transfected with plasmids expressing Pr55^Gag/Pr55^Gag-eGFP or with a replication-competent HIV provirus plasmid pNL4.3 and a Pr55^Gag-eGFP-expressing plasmid. One day after transfection, the PM of the cells were stained with the dSQ12S probe, and living cells were imaged by confocal microscopy. (A) Effect of PIP5K1 isoform silencing on the distribution of Pr55^Gag in transfected cells. Representative z-stack images (green for Pr55^Gag, red for the dSQ12S probe, and yellow for the merged channels) for cells transfected with nontargeting control siRNA or siRNA targeting PIP5K1α, PIP5K1β, or PIP5K1γ (a, b, c, and d, respectively) are shown. (B) The colocalization of Pr55^Gag with the PM probe (dSQ12S) was quantified with ImageJ software. Each dot represents the average of R for 10 z-stack images of a cell. Horizontal black bars stand for the mean value. (C) Effect of PIP5K1 isoforms silencing on the distribution of Pr55^Gag produced by the replication-competent HIV-1_NL4.3 in transfected cells. Representative z-stack images (green for Pr55^Gag, red for the dSQ12S probe, and yellow for the merged channels) from cells transfected with nontargeting control siRNA or siRNAs targeting PIP5K1α, PIP5K1β, or PIP5K1γ (a, b, c, and d, respectively) are shown. (D) The colocalization of Pr55^Gag produced by the replication-competent HIV-1_NL4.3 with the PM probe dSQ12S was quantified with ImageJ software. Each dot represents the average of R for 10 z-stack images of a cell. Horizontal black bars stand for the mean value. Mann-Whitney tests were performed to assess the significance of differences. Significant results are indicated (****, P < 0.0001; ns, not significant).