1. Volume 7, Issue 6, Pages 1245-1254 (June 2001)
Cytoplasmic Recruitment of INI1 and PML on Incoming HIV Preintegration Complexes 
Priscilla Turelli, Vassilis Doucas, Errol Craig, Bastien Mangeat, Natacha Klages, Ronald Evans, Ganjam Kalpana, Didier Trono 
Molecular Cell 
Volume 7, Issue 6, Pages (June 2001)
DOI: /S (01)

2. Figure 1 HIV-1-Induced Cytoplasmic Redistribution of INI1 and PML
(A) Control (panels a–c) or GFP-INI1-transfected (panels d–i) CD4+ HeLa cells were either mock-treated (panels a, d, and g) or infected with wild-type (WT; panels b, e, and h) or ΔEnv (panels c, f, and i) HIV-1 and fixed 2 hr postinfection. Endogenous PML (red) was detected with a rabbit polyclonal antiserum and an alexa-568-conjugated secondary antibody (panels a–c and g–i). The nuclei were stained with DAPI (blue). The overlap of red (PML) and green (GFP-INI1) signals results in a yellow color.
(B) HeLa cells were either mock-treated (panels a, c, e, g, and i) or infected with wild-type HIV-1 at a m.o.i. of 10 (panels b, d, f, and h) or 0.3 (panel j), and examined by microscopy 2 hr later. Panels a and b: cells expressing GFP-NLS and stained with DAPI; panels c and d: cells transfected with a GFP-Rev vector; panels e and f: cells analyzed by indirect immunofluorescence for the detection of endogenous INI1 protein; panels g and h: cells transfected with GFP-INI1 and stained for Brahma (red); and panels i and j: cells stained for endogenous PML; the same proportion of GFP-INI1-expressing cells exhibited cytoplasmic relocalization of this protein (data not shown)
Molecular Cell 2001 7, DOI: ( /S (01) )

3. Figure 2 Kinetics of INI1 and PML Redistribution
GFP-INI1-transfected CD4+ HeLa cells were mock-treated (A) or HIV-1-infected (B–E) and fixed with methanol at 30 min (B), 2 hr (C), 4 hr (D), and 6 hr (E) postinoculation. PML (red) was detected by indirect immunofluorescence using a polyclonal antiserum. Images were recorded by confocal microscopy. The left panels correspond to images obtained with the red filter, and the central panels to those with the green filter. The right panels represent merged images. The overlap of red (PML) and green (GFP-INI1) signals results in a yellow color
Molecular Cell 2001 7, DOI: ( /S (01) )

4. Figure 3
Exportin Dependence of HIV-Induced INI1 and PML Redistribution
GFP-INI1-transfected CD4+ HeLa cells were mock- or leptomycin B (LMB)-treated for 30 min prior to infection with HIV-1. PML (red) was detected by indirect immunofluorescence with a rabbit anti-PML polyclonal antibody while GFP-INI1 yields a green signal. The nuclei (blue) were stained with DAPI. The cells were examined with an Axiophot microscope
Molecular Cell 2001 7, DOI: ( /S (01) )

5. Figure 4 Viral Requirements of INI1 and PML Cytoplasmic Recruitment
GFP-INI1-transfected CD4+ HeLa cells were either mock-treated (panel a) or infected with wild-type HIV-1 (panel b), VSV G-pseudotyped HIV-1 (panel c), VSV G-coated HIV-1 vector particles produced from a multiply deleted packaging construct with (panel d) or without (panel e) packageable RNA, VSV G-pseudotyped MLV vector particles (panel f), or adenovirus (panel g). Cells fixed 2 hr postinfection were analyzed by indirect immunofluorescence and confocal microscopy. PML is red, GFP-INI1 is green, and the colocalization of the two proteins results in a yellow signal
Molecular Cell 2001 7, DOI: ( /S (01) )

6. Figure 5 INI1 and PML Redistribution in HIV-1-Infected Primary Cells
PHA/IL-2-activated PBLs (A) or monocyte-derived macrophages (B) were either mock-treated (top panels) or infected with VSV G-pseudotyped ΔEnv HIV-1 particles (bottom panels), and analyzed 2 hr later by indirect immunofluorescence and confocal microscopy, using monoclonal anti-PML and polyclonal anti-INI1 antibodies. Endogenous PML is in red, endogenous INI1 in green. The nuclei of macrophages are outlined in images obtained in contrast phase ([B], right side)
Molecular Cell 2001 7, DOI: ( /S (01) )

7. Figure 6 Visualization of HIV-1 Preintegration Complexes
(A) Particles labeled by the incorporation of GFP-WXXF and endogenous reverse transcription with rhodamine-conjugated dUTP were used to infect 293T cells. Cytoplasmic extracts were harvested at 2.5 hr postinfection and loaded on a sucrose gradient in parallel with purified virions. The fractions were analyzed for refraction index, RT activity, and p24 content, as well as for GFP reactivity by Western blot.
(B) HeLa cells infected with dually labeled virions at an m.o.i. of 30 were analyzed by confocal microscopy 2.5 hr postinfection. The overlay in the left panel reveals the colocalization of the rhodamine and GFP signals, while the right panel shows the contrast phase analysis
Molecular Cell 2001 7, DOI: ( /S (01) )

8. Figure 7 Colocalization of INI1 with the Incoming HIV-1 Genome
CD4+ HeLa cells were transfected (panels a, b, and d–f) or not (c) with a GFP-INI1-expressing plasmid. Two days later, cells were infected as follows: panel a, heat-inactivated rhodamine-labeled wild-type HIV-1; panel b, nonlabeled wild-type HIV-1; panels c and d, rhodamine-labeled wild-type HIV-1; panel e, rhodamine-labeled VSV G-pseudotyped Gag-Pol-based HIV-1 vector particles; and panel f, VSV G-pseudotyped Gag-Pol-based, genomeless, vector particles
Molecular Cell 2001 7, DOI: ( /S (01) )

9. Figure 8
Interference with the Postentry Preintegration Steps of HIV Infection
(A) GFP-INI-transfected CD4+ HeLa cells were incubated with an arsenic-containing medium (4 μM) for 16 hr before mock (panel a) or HIV (panel b) infection. Cells fixed 2 hr postinfection were analyzed by indirect immunofluorescence. Endogenous PML (red) was detected with a rabbit polyclonal antiserum. The nuclei were stained with DAPI (blue).
(B) Left panel: HeLa cells, pretreated or not with arsenic at indicated concentrations, were infected with a GFP-expressing HIV vector at different m.o.i. and subjected to FACS analysis 5 days later to score for transduction efficiency. Right panel: the same experiment in 293T cells transfected with a control or a PML-expressing vector. Transduction efficiency in the absence of arsenic was given the arbitrary value of 1.
(C) PCR-based semiquantitative measurement of actin (top panel) or vector (bottom panels) DNA, in 5-fold serial dilutions of extracts from HeLa cells infected in (B) (top panels) or of vector plasmid DNA (bottom panel)
Molecular Cell 2001 7, DOI: ( /S (01) )