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. 2021 Jan 13;95(3):e00883-20. doi: 10.1128/JVI.00883-20

FIG 6.

FIG 6

The biotinylated antiretroviral analog of PAV-206 colocalizes with HIV-1 Gag in situ. (A) Schematic of the PLA approach for detecting colocalization of compound with Gag. 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 incubating with primary antibodies (rabbit anti-biotin and mouse anti-Gag), followed by PLA secondary antibodies (anti-rabbit IgG coupled to [+] PLA oligonucleotide and anti-mouse IgG coupled to [–] PLA oligonucleotide). The addition of other PLA reagents leads to connector oligonucleotides linking the “+” and “–” oligonucleotides only if the primary antibodies are colocalized; this in turn results in the PLA amplification reaction. The addition of an oligonucleotide that recognizes a sequence in the amplified regions and is coupled to a red fluorophore (red star) results in intense spots at sites where biotinylated compound and Gag are colocalized in situ. After PLA, IF was performed by adding secondary antibody conjugated to a green fluorophore (green star) to detect any unoccupied Gag antibody, thus marking Gag-expressing cells with low-level green fluorescence. (B) Graph showing the average number of biotin-Gag PLA spots per cell for each condition, with “+” indicating HIV-1-infected cells and “–” indicating uninfected cells. Twenty fields were analyzed for each group (containing a total of 186 to 316 cells per group), with error bars showing SEM. *, Significant difference in the number of biotin-Gag 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-Gag PLA spots alone in grayscale. To the right are the same fields shown as a merge of three color channels: biotin-Gag PLA (red), Gag IF (green), and DAPI-stained nuclei (blue). Scale bars, 10 μm.