Journal: Journal of virology

Article Title: Sustained small interfering RNA-mediated human immunodeficiency virus type 1 inhibition in primary macrophages.

doi: 10.1128/jvi.77.13.7174-7181.2003

Figure Lengend Snippet: FIG. 2. CCR5 and p24 siRNAs inhibit HIVBAL infection in MDMs. (a) MDMs were transfected with the indicated doses of CCR5 (I) or p24 () siRNA and infected 2 days later with HIVBAL. Cell-free virus production was measured on day 7 postinfection by p24 ELISA. (b) MDMs were either mock transfected () or transfected with the GFP (I), p24 (Œ), or CCR5 () siRNA or with the p24 and CCR5 siRNAs (*) and infected after 2 days with HIVBAL, and virus production was measured by p24 ELISA at the indicated times postinfection. (c) The siRNA-transfected cells described in panel b were stained with anti-p24–FITC 15 days after infection and examined by flow cytometry. The percentage of p24 cells is shown in each panel. (d) siRNA-transfected and HIVBAL-infected MDMs were probed for HIV-1 RNA by in situ hybridization with a fluorescein-labeled HIV-1 gag-pol oligonucleotide probe cocktail 7 days after infection. Fluorescence microscopy (magnification, 200) was used to evaluate fluorescence signals for HIV-1 RNA (bottom). At the top are the same cells counterstained with Texas red-X phalloidin.

Article Snippet: To test CCR5 expression and HIV-1 infection, adherent MDMs were trypsinized at the times indicated and stained with biotin-conjugated CCR5 antibody (R&D Systems, Inc., Minneapolis, Minn.), followed by avidin-labeled streptavidin-phycoerythrin (BD Pharmingen, San Diego, Calif.).

Techniques: Infection, Transfection, Virus, Enzyme-linked Immunosorbent Assay, Staining, Cytometry, RNA In Situ Hybridization, Labeling, Fluorescence, Microscopy

Journal: Journal of virology

Article Title: Sustained small interfering RNA-mediated human immunodeficiency virus type 1 inhibition in primary macrophages.

doi: 10.1128/jvi.77.13.7174-7181.2003

Figure Lengend Snippet: FIG. 3. CCR5, but not p24, siRNA persists in uninfected MDMs. (a) Modified Northern blot analysis showing levels of internalized CCR5 and p24 siRNAs in MDMs on the indicated days after transfection. Before loading, samples were normalized for total RNA content. The sense strand of each siRNA was end labeled with -32P and used as a probe. Lanes loaded with graded amounts of the antisense strand of siRNA and mock-transfected samples served as positive and negative controls, respectively. (b) CCR5 (top) and GFP (bottom) siRNA-transfected MDMs were examined for CCR5 expression over time. Overlay histograms of CCR5-stained mock-transfected (open solid line), control immunoglobulin- stained (open dotted line), and siRNA-transfected (filled) cells are shown. (c) RT-PCR for CCR5 and -actin mRNA expression was performed with mock-transfected (lanes 2 to 5) and CCR5 siRNA-transfected (lanes 6 to 9) cells on days 1 (lanes 2 and 6), 4 (lanes 3 and 7), 7 (lanes 4 and 8), and 15 (lanes 5 and 9) after transfection (M, molecular weight marker; lane 1, negative control). CCR5 mRNA was not detected, even after an additional 25 cycles of PCR amplification (data not shown).

Article Snippet: To test CCR5 expression and HIV-1 infection, adherent MDMs were trypsinized at the times indicated and stained with biotin-conjugated CCR5 antibody (R&D Systems, Inc., Minneapolis, Minn.), followed by avidin-labeled streptavidin-phycoerythrin (BD Pharmingen, San Diego, Calif.).

Techniques: Northern Blot, Transfection, Labeling, Expressing, Staining, Control, Reverse Transcription Polymerase Chain Reaction, Molecular Weight, Marker, Negative Control

Journal: Journal of virology

Article Title: Sustained small interfering RNA-mediated human immunodeficiency virus type 1 inhibition in primary macrophages.

doi: 10.1128/jvi.77.13.7174-7181.2003

Figure Lengend Snippet: FIG. 4. CCR5, but not p24, siRNA confers sustained and uniform protection when MDMs are infected at increasing intervals after transfection. MDMs were transfected with GFP (top), p24 (middle), or CCR5 (bottom) siRNA and infected with HIVBAL at the indicated times after transfection. Cells were analyzed 10 days postinfection for p24 expression by flow cytometry. The percentage of p24 cells is shown in each panel.

Article Snippet: To test CCR5 expression and HIV-1 infection, adherent MDMs were trypsinized at the times indicated and stained with biotin-conjugated CCR5 antibody (R&D Systems, Inc., Minneapolis, Minn.), followed by avidin-labeled streptavidin-phycoerythrin (BD Pharmingen, San Diego, Calif.).

Techniques: Infection, Transfection, Expressing, Cytometry

Journal: Journal of virology

Article Title: Sustained small interfering RNA-mediated human immunodeficiency virus type 1 inhibition in primary macrophages.

doi: 10.1128/jvi.77.13.7174-7181.2003

Figure Lengend Snippet: FIG. 5. p24, but not CCR5, siRNA suppresses HIV-1 replication in an established infection. (a) MDMs infected with HIVBAL for 16 days (90% of the MDMs were p24; data not shown) were transfected with CCR5 siRNA and examined for p24 expression 3 days later. The percentage of p24 cells is shown in each panel. (b) MDMs infected for 16 days were transfected with p24 or control siRNA and examined for p24 expression on various days posttransfection.

Article Snippet: To test CCR5 expression and HIV-1 infection, adherent MDMs were trypsinized at the times indicated and stained with biotin-conjugated CCR5 antibody (R&D Systems, Inc., Minneapolis, Minn.), followed by avidin-labeled streptavidin-phycoerythrin (BD Pharmingen, San Diego, Calif.).

Techniques: Infection, Transfection, Expressing, Control