Figure 4

HIV-1 Nef induces loss of F-actin assembly and inhibits retinoid receptor-mediated transcription. A) Jurkat cells were transfected for 36 h with 5.0 ug of Nef/WT or Nef/G2A plasmids using Fugene-HD. The cells were fixed, permeabilized, and blocked with BSA as described in Experimental Procedures. Cells were incubated with anti-Nef anti-serum (1:500 in 5% BSA/PBS) for 1-2 h and labeled with FITC-conjugated secondary antibody. Cells were stained for F-actin using Alexa Fluor 555 Phalloidin and mounted in Prolong Gold Antifade Reagent with DAPI. Confocal images were obtained using an Olympus Fluoview 1000-inverted microscope. Each figure is a quadron for DAPI, Nef, F-actin, and merged images. F-actin stained Nef expressing cells are indicated by arrows. Bar, 2.5 um. B-E) Jurkat (B, E) or PBT (C) cells were transfected with 5.0 ug TATADR1-Luc or TKCRBPII-Luc plasmids respectively in the presence of 2.5 ug of indicated plasmids. After 16 h, cells were treated as indicated and luciferase activity measured 24 h later as described in Materials and Methods. D) Jurkat cells were transfected with 2.5 ug of SP1-Luc in the presence of indicated plasmids and luciferase activity measured 36 h later. F) Jurkat cells were transfected with 5.0 ug TATADR1-Luc in the presence of 2.5 ug of indicated plasmids. After 16 h, cells were treated with indicated compounds and luciferase activity measured 24 h later as described in Experimental Procedures.