Schematic representation of the screen. Arrayed pools of siRNAs were transfected into TZM-bl cells in a 384-well format. After 72 hours, HIV-IIIB virus was added, and 48 hours after infection, cultured supernatant was removed and added to fresh TZM-bl cells. In part one, 48 hours after infection, the siRNA transfected cells were fixed, permeabilized, stained, and imaged for HIV p24 protein and DNA. In part two, cells were cultured for 24 hours after the addition of supernatant, then analysed, exposed to a luminescent b-Gal substrate, and relative light units (RLU) recorded. From Fig. 1A, Brass et al. Science, 2008. 

 

The results of the screen are shown with the siRNA SMARTpools ranked in order of average Z score, from lowest (decreased infection) to highest (increased infection). The position of known influenza A virus-host factors and several newly identified genes from the screen are indicated. From Fig. 1C, Brass et al. Cell, 2009.

Immunofluorescence images showing the block to HIV infection with loss of TNPO3. Cells were treated as described in (B), with either the luciferase (Luc), negative control siRNA; or TNPO3, siRNA #8 targeting TNPO3. “Merge” denotes the combined image for nuclei (blue) and HIV p24 (green). From Fig. 3H, Brass et al. Science, 2008.

A549 or U2OS cells stably overexpressing IFITM3 protein or vector alone were infected with influenza A H1N1 WSN/33. Twelve hours later, cells were fixed and stained for surface HA expression. Values represent the mean ± SD, n = 3 (green: HA, blue: nuclei; 43). From Fig. 4C, Brass et al. Cell, 2009.

Localization of IFITM3 in A549 cells stably overexpressing IFITM3 protein, by confocal microscopy