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. 2013 Apr 27;41(12):6286–6299. doi: 10.1093/nar/gkt306

Figure 2.

Figure 2.

HIV-1 gRNA and DDX3 are localized in large cytoplasmic RNA granules (A) HeLa cells were transfected with 0.5 μg of pNL4-3 and 0.2 μg of pCIneo-HA-DDX3 for 24 h and prepared for LSCM as indicated in ‘Materials and Methods’ section. The HIV-1 gRNA (green staining) and DDX3 (red staining) were observed either disperse throughout the cytoplasm (left, scale bar 25 μm) or co-localizing in large cytoplasmic granules (right, scale bar 10 μm). White arrowheads indicate cytoplasmic granules. Co-localized points are indicated in white. Merge images include nucleus staining with Hoechst (blue). Colour profiles from a region of interest (ROI) are also shown below. (B) HeLa cells were transfected with 0.5 μg of pNL4-3 and 0.2 μg of pCIneo-HA-CRM1 for 24 h and prepared for LSCM. Green staining corresponds to the gRNA, and red staining identifies the CRM1 protein. White arrowheads indicate HIV-1 gRNA granules. Merge images include nucleus staining with Hoechst (blue). Scale bar 25 μm. (C) Control (siCtrl) or DDX3-depleted (siDDX3) HeLa cells were transfected with 0.3 μg of pNL4-3R or pNL4-3R ΔRev proviral DNA, and gRNA translation (Gag–Renilla luciferase activity normalized to the amount of cytoplasmic gRNA as measured by RT–qPCR) was determined as described in ‘Materials and Methods’ section. Results were normalized to siCtrl (set to 100%) and are presented as mean ± SD of three independent experiments. **P < 0.01; ***P < 0.001 (non-directional t-test). (D) HeLa cells were transfected with 0.5 μg of pNL4-3 Gag-Stop and 0.2 μg of pCIneo-HA-DDX3 for 24 h and prepared for LSCM as indicated in ‘Materials and Methods’ section. Green staining corresponds to the gRNA, and red staining identifies DDX3. White arrowheads indicate cytoplasmic granules. Co-localized points are indicated in white. Merge image includes nucleus staining with Hoechst (blue). Scale bar 25 μm.