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. Author manuscript; available in PMC: 2009 Apr 17.
Published in final edited form as: Cell Host Microbe. 2008 Mar 13;3(4):245–252. doi: 10.1016/j.chom.2008.03.001

Figure 3. Reduction of constitutive BST-2-expression in HeLa cells decreases the virologic phenotype of vpu.

Figure 3

A) HeLa cells were transfected to express transiently either no short-hairpin (sh) RNA (“empty vector”) or various shRNAs targeting different sequences in BST-2 mRNA (“TI-1”, “TI-3”, or “TI–4”), along with GFP encoded on a separate plasmid, then stained three days later for surface BST-2/CD317 and analyzed by two-color flow cytometry. Horizontal and vertical lines indicate gates set using either GFP-negative cells or a primary antibody isotype control for BST-2; colors are arbitrary and distinguish GFP-negative, -low positive, and -high positive cells. Total DNA in each transfection was 1.6 μg, 1.4 of which was the shRNA expression vector. B) HeLa cells were transfected to express either no shRNA or shRNAs TI-1, -3, -4, or an equimolar mixture of TI-1, -3, and –4, along with wild-type or vpu-negative full length viral DNA at a weight ratio of 2:1:: shRNA-vector: proviral plasmid. A CXCR4 receptor blocker (AMD3100) was used to limit viral production to the initially transfected cells, and the fraction of the total p24 capsid antigen produced that was secreted into the media was measured two days later.