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. Author manuscript; available in PMC: 2012 Dec 27.
Published in final edited form as: Cell Rep. 2012 Oct 4;2(4):807–816. doi: 10.1016/j.celrep.2012.09.008

Fig. 1. BRD4 depletion or inhibition increases HIV-1 replication.

Fig. 1

(A, B) The results of the indicated RNAi library screens with the siRNA pools ranked in order of average relative fold infection, from lowest to highest. The positions of known HDFs as well as the newly identified HIV-1-competitive factor (HCF), BRD4, from the screen are indicated. (a) Ambion Library; (b) esiRNA Library

(C) HeLa MAGI cells were transfected with the indicated siRNAs for 72 hr, then infected with HIV-IIIB (Part I). Forty-eight hr later the viral supernatant was transferred to a new plate of HeLa MAGI cells for 48 hr (Part II). Part I and II cells were stained for p24. Relative fold infection is normalized to the non-targeting control siRNA (Con). Identically transfected cells were assessed for BRD4 mRNA knockdown using quantitative PCR (qPCR) and normalized to the control siRNA (right).

(D) Cells were treated as in (C) but were transfected with esiRNA pools as indicated. Values were normalized to the non-targeting firefly luciferase (FLuc) esiRNA control pool. Identically transfected cells were assessed for BRD4 mRNA knockdown using quantitative PCR (qPCR) and normalized to the control FLuc esiRNA pool (right).

(E) Jurkat T cells were stably transduced with the indicated shRNAs then infected with VSV-G NL4-3-GFP HIV-1. At 72 hr post infection the percentage of GFP positive cells was determined by flow cytometry, and the relative fold infection normalized to the shFLuc-expressing control line.

(F) Western blot for cells in (E). kDa = kilodaltons

(G) Primary Human CD4+ T cells were stably transduced with the indicated shRNAs then infected with VSV-G NL4-3-GFP HIV-1. At 72 hr post infection the percentage of GFP positive cells was determined by flow cytometry, and the relative fold infection normalized to the shFLuc-expressing control line.

(H) Western blot for cells in (G). The levels of a non-specific band (loading) show relative protein loading.

(I) HeLa MAGI cells were treated with either DMSO or JQ1 (500 nM) for 1 hr, then infected with HIV-IIIB as in (C).

(J) Jurkat T cells were treated with either DMSO or JQ1 (500 nM) for 1 hr, then infected with VSV-G NL4-3-GFP HIV-1 using the indicated multiplicity of infections (M.O.I). After 48 hr the percentage of GFP positive cells was determined by flow cytometry.

Values represent the mean +/− S.E.M. (standard error), N > 3 throughout. * P < 0.05, ** P < 0.01. Results were analyzed by unpaired t tests. See Fig. S1.