Journal:

Article Title: Regulation of Human Immunodeficiency Virus Type 1 Infectivity by the ERK Mitogen-Activated Protein Kinase Signaling Pathway

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Figure Lengend Snippet: Inhibition of HIV-1 infectivity by a MAPK antisense oligonucleotide (AS) or scrambled control oligonucleotide (SC). (A) Depletion of p44/42 MAPK expression by AS. HeLa cells were treated with AS or SC at the indicated concentrations. HeLa cells were grown to 80% confluence in Dulbecco’s minimal essential medium (DMEM) containing 10% FCS in 35-mm-diameter six-well plates, and washed twice with serum-free DMEM. Appropriate concentrations of oligonucleotides in 125 μl of serum-free DMEM were preincubated at room temperature for 15 min with 125 μl of serum-free DMEM containing 40 μg of DOTMA (Lipofectin) (Gibco-BRL) per ml. This solution was then mixed with an additional 250 μl of serum-free DMEM. Cells were incubated with the mixture for 6 h at 37°C in the presence of 5% CO2. Subsequently, the medium containing DOTMA was removed and the incubation was continued for 48 h in fresh medium containing 10% FCS. After 48 h incubation, MAPK expression was determined by immunoblotting of equivalent amounts of total protein with anti-MAPK (anti-ERK1 [p44] and anti-ERK2 [p42]). (B) HeLa cells were transfected with 3 μg of pHXB2 containing HIV-1 proviral DNA following depletion of MAPK by treatment with MAPK antisense oligonucleotide as described for panel A. Virus production was determined by quantitation of RT activity in the supernatants of the transfected HeLa cell cultures at 48 h after transfection. Virus infectivity was determined by infection of CEM cells and quantitation of RT activity in the supernatants of the newly infected cells on day 7 after infection. (C) Treatment with AS does not inhibit expression of an HIV-1 LTR CAT reporter plasmid transactivated by coexpression of HIV-1 Tat. HeLa cells were treated with the oligonucleotides at 0.2 μM and cotransfected with 0.5 μg of pUIIIRCAT and 0 or 0.5 μg of the HIV-1 Tat expressor plasmid pSVLtat by the same method as described for panels A and B. Shown is CAT activity in the transfected cell lysate at 48 h posttransfection, as determined by the conversion of chloramphenicol to its acetylated forms.

Article Snippet: Appropriate concentrations of oligonucleotides in 125 μl of serum-free DMEM were preincubated at room temperature for 15 min with 125 μl of serum-free DMEM containing 40 μg of DOTMA (Lipofectin) (Gibco-BRL) per ml.

Techniques: Inhibition, Infection, Expressing, Incubation, Western Blot, Transfection, Quantitation Assay, Activity Assay, Plasmid Preparation