Journal: PLOS Biology

Article Title: DNA damage induced by HIV-1 Vpr triggers epigenetic remodeling and transcriptional programs to enhance virus transcription and latency reactivation

doi: 10.1371/journal.pbio.3003621

Figure Lengend Snippet: (A) Electrostatic surface potential of a Vpr-DCAF1 co-complex. Surfaces associated with hyperactive or DDR-deficient Vpr mutants are colored in blue and red, respectively. (B, C) Immunofluorescence microscopy quantification of DDR activation following infection of HeLa or differentiated THP1 cells with Vpr WT or mutant viruses 48 hours post-infection ( n = 50 cells). Dashed boxes highlight hyperactive and DDR-deficient Vpr mutants associated with sub-surfaces colored blue and red, respectively, from . The data underlying this Figure can be found in . (D) Immunoblot analysis of histone marks in HeLa cells infected with indicated viruses 48 hours post-infection. The unmodified images underlying this Figure can be found in . (E) Circle graphs displaying amino acid variance at relevant positions identified in the mutagenesis screen with consensus residues in the middle of the circle. Sequences obtained from Los Alamos database ( n ≈ 4,100). (F) Immunofluorescence microscopy quantification of DDR activation and histone marks following infection of HeLa cells with Vpr WT , Vpr Y15R , or Vpr Y15H mutant viruses 48 hours post-infection ( n = 50 cells). Analyses performed using a one-way ANOVA; ns, not significant; *** p < 0.001; ** p < 0.01; * p < 0.05. The data underlying this Figure can be found in .

Article Snippet: For coupling immunofluorescence microscopy with bimolecular fluorescence complementation, 48-hours post-transfection HeLa cells were fixed with 4% PFA, and immunofluorescence labeling was performed as described above (DCAF1, 1:400, Proteintech 11612-1-AP; GFP, 1:500, Cell Signaling 2956).

Techniques: Immunofluorescence, Microscopy, Activation Assay, Infection, Mutagenesis, Western Blot

Journal: PLOS Biology

Article Title: DNA damage induced by HIV-1 Vpr triggers epigenetic remodeling and transcriptional programs to enhance virus transcription and latency reactivation

doi: 10.1371/journal.pbio.3003621

Figure Lengend Snippet: (A) Immunofluorescence microscopy quantification of histone marks in HeLa and differentiated THP1 cells infected with indicated viruses 48 hours post-infection ( n = 50 cells). Analyses performed using a one-way ANOVA; ns, not significant; *** p < 0.001. The data underlying this Figure can be found in . (B) Immunofluorescence microscopy quantification of histone marks following infection of HeLa cells expressing control or DCAF1 knockdown constructs 48 hours post-infection ( n = 50 cells). HeLa cells were sequentially transfected and infected with the indicated provirus and a shRNA construct that expresses a BFP reporter from a separate promoter. mCherry and BFP expressing cells were validated for loss of DCAF protein and changes to histone marks. Analyses performed using a one-way ANOVA; ns, not significant; *** p < 0.001. The data underlying this Figure can be found in . (C) Representative fluorescence microscopy images and quantification of DCAF1 localization in HeLa, MDMs, and differentiated THP1 cells infected with the indicated viruses 48 hours post-infection ( n = 50 cells). Analyses performed using a one-way ANOVA; ns, not significant; *** p < 0.001. The data underlying this Figure can be found in .

Article Snippet: For coupling immunofluorescence microscopy with bimolecular fluorescence complementation, 48-hours post-transfection HeLa cells were fixed with 4% PFA, and immunofluorescence labeling was performed as described above (DCAF1, 1:400, Proteintech 11612-1-AP; GFP, 1:500, Cell Signaling 2956).

Techniques: Immunofluorescence, Microscopy, Infection, Expressing, Control, Knockdown, Construct, Transfection, shRNA, Fluorescence

Journal: PLOS Biology

Article Title: DNA damage induced by HIV-1 Vpr triggers epigenetic remodeling and transcriptional programs to enhance virus transcription and latency reactivation

doi: 10.1371/journal.pbio.3003621

Figure Lengend Snippet: (A, B) Representative live cell fluorescence microscopy images of HeLa or differentiated THP1 cells expressing indicated constructs 48 hours post-transfection or -infection, respectively. For washout experiments, cells were either untreated or incubated with detergent for 15 min prior to live-cell imaging. (C) Live-cell fluorescence quantification of dual or singly fluorescence-positive cells from and ( n = 50 cells). The data underlying this Figure can be found in . (D) Diagrams of BiFC co-transfections and representative live cell fluorescence microscopy images of HeLa cells co-expressing indicated Vpr constructs. (E) Representative fluorescence microscopy images of Vpr (left) and DCAF1 (right) localization in HeLa cells infected with indicated viruses with quantification of DCAF1 cytoplasmic-to-nuclear ratio (Top) and induction of γH2A.X foci (Bottom) ( n = 45 cells). Cells were co-transfected with NTvenus-Vpr and CTvenus-Vpr constructs for 24 hours prior to live-cell imaging. Analyses performed using a one-way ANOVA; ns, not significant; *** p < 0.001. The data underlying this Figure can be found in .

Article Snippet: For coupling immunofluorescence microscopy with bimolecular fluorescence complementation, 48-hours post-transfection HeLa cells were fixed with 4% PFA, and immunofluorescence labeling was performed as described above (DCAF1, 1:400, Proteintech 11612-1-AP; GFP, 1:500, Cell Signaling 2956).

Techniques: Fluorescence, Microscopy, Expressing, Construct, Transfection, Infection, Incubation, Live Cell Imaging