FIG 4.

The silencing of PIP5K1α and PIP5K1γ has different effects on the retargeting of HIV-1 Pr55^Gag to endosomal pathways. (A) Effect of the silencing of PIP5K1 isoforms on the rerouting of Pr55^Gag to early endosomes. TZM-bl HeLa cells, transfected with either a nontargeting control siRNA or with siRNAs targeting each PIP5K1 isoform, were then transfected with plasmids encoding Pr55^Gag/Pr55^Gag-eGFP and mCherry-Rab5. Z-stack confocal images were acquired from living cells as previously described. Representative yellow merged images (green for Pr55^Gag and red for mCherry-Rab5) are shown for cells transfected with nontargeting control siRNA or siRNA targeting PIP5K1α, PIP5K1β, or PIP5K1γ (a, b, c, and d, respectively). (B) The colocalization of Pr55^Gag-eGFP with mCherry-Rab5 was quantified with ImageJ software. Each dot represents the average of R for 10 z-stack images of a representative cell. Horizontal black bars stand for the mean value. (C) Effect of the silencing of PIP5K1 isoforms on the rerouting of Pr55^Gag to late endosomes. TZM-bl HeLa cells, transfected with either a nontargeting control siRNA or with siRNAs targeting each PIP5K1 isoform, were then transfected with plasmids encoding Pr55^Gag/Pr55^Gag-eGFP and mCherry-Rab7A. Z-stack confocal images were acquired from living cells as previously described. Representative yellow merged images (green for Pr55^Gag and red for mCherry-Rab7A) are shown for cells transfected with nontargeting control siRNA or siRNA targeting PIP5K1α, PIP5K1β, or PIP5K1γ (a, b, c, and d, respectively). (D) The colocalization of Pr55^Gag-eGFP with mCherry-Rab7A was quantified with ImageJ software. Each dot represents the average of R for 10 z-stack images of a representative cell. Horizontal black bars stand for the mean value. (E) Effect of PIP5K1 isoform silencing on the relocalization of Pr55^Gag to acidic vesicles. TZM-bl HeLa cells, transfected with either a nontargeting control siRNA or with siRNAs targeting each PIP5K1 isoform, were cotransfected with Pr55^Gag- and Pr55^Gag-mCherry-expressing plasmids. Lysosomes were stained in green with the LysoTracker probe 24 h after DNA transfection. Acquisitions of z-stack confocal images were performed as previously described, and the colors of Pr55^Gag-mCherry and the green LysoTracker probe were inversed to prevent confusion. Representative yellow merged images (green for Pr55^Gag and red for the LysoTracker probe) are shown for cells transfected with nontargeting control siRNA or siRNA targeting PIP5K1α, PIP5K1β, or PIP5K1γ (a, b, c, and d, respectively). (F) The colocalization of Pr55^Gag-mCherry with the green LysoTracker probe was quantified with ImageJ software. Each dot represents the average of R for 10 z-stack images of a representative 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; ***, 0.0001 < P < 0.001; ns, not significant).