Figure 6. Effect of berberine on HIV PI-induced cytoplasmic translocation of RNA binding protein HuR in mouse macrophages.

Mouse J774A.1 cells were pretreated with various amounts of berberine (BBR, 0, 0.5, 1, 2 µg/ml) for 2 h and then treated with atazanavir (ATZV, 15 µM) or lopinavir (LOPV, 15 µM) for 24 h. The nuclear proteins and cytoplasmic proteins were isolated as described in “Methods”. Three independent experiments were performed. Representative immunoblots against HuR from (A) nuclear proteins; (B) cytoplasmic proteins; (C) total cellular proteins. Lamin B and β-actin were used as loading controls for nuclear proteins and cytosolic/total cellular proteins. The density of the immunoreactive bands was analyzed using Image J software and normalized to lamin B or β-actin control. D. Immunofluorescent staining of cellular distribution of HuR. Cells were pretreated with berberine (BBR, 0.5 µg/ml) for 2 h and then treated with vehicle control DMSO, atazanavir (ATZV, 15 µM) or lopinavir (LOPV, 15 µM) for 24 h. Expression of HuR was detected with mouse monoclonal antibody against HuR and donkey anti-mouse IgG conjugated with Alex Fluor-594. Nuclei were stained with DAPI. HuR expression was visualized under a confocal fluorescence microscope with a 40× objective using a dual filter set for DAPI and rhodamine. Three experiments were performed that showed similar results.