FIG 11.

The biotinylated antiretroviral PAV-206 analog colocalizes in situ with DDX6, an RNA granule protein in assembly intermediates that colocalizes with ABCE1. (A) Schematic of the PLA approach for detecting colocalization of compound with DDX6. 293T cells chronically infected with HIV-1 (293T-HIV) or uninfected 293T cells were treated with indicated amounts of PAV-818 (the biotinylated active compound), PAV-543 (the biotinylated inactive compound), or DMSO for 16 h. PLA was performed by incubation with primary antibodies (mouse anti-biotin and rabbit anti-DDX6), followed by PLA secondary antibodies and other reagents as described in Fig. 6. Red spots indicate sites where biotinylated compound and DDX6 are colocalized in situ. After PLA, IF was performed (green star) to mark intracellular DDX6 with low-level green fluorescence. (B) The graph shows the average number of biotin-DDX6 PLA spots per cell for each condition, with “+” indicating HIV-1-infected cells and “–” indicating uninfected cells. Five fields were analyzed for each group (containing a total of 39 to 73 cells per group), with error bars showing the SEM. *, Significant difference in the number of biotin-DDX6 PLA spots per cell when comparing treatment with PAV-818 versus PAV-543, both at 10 μM (P < 0.001). (C) A representative field for each group quantified in panel B is shown, except for DMSO treatment. Fields on the left show biotin-DDX6 PLA spots alone in grayscale. To the right are the same fields shown as a merge of three color channels: biotin-DDX6 PLA (red), DDX6 IF (green), and DAPI-stained nuclei (blue). Scale bars, 10 μm.