Viral Replication and Cell Viability assays in the presence of exogenous p16INK4A treatment. A) J1.1 and U1 latently infected HIV-1 cell lines were treated with GST or GST-p16INK4A (0.1 μg or 0.5 μg). Cell culture supernatants were collected at 48 hours post treatment and assayed for reverse transcriptase (RT) activity measured in cpm. B) CEM, Jurkat, H9, and U937 uninfected cell lines were treated with GST or GST-p16INK4A (0.1 μg or 0.5 μg). Fourty-eight hours post treatment, the cells were measured for viability with MTT reagent. C) Jurkat uninfected T cells were treated with an excess (2.5 μg) of GST or GST-p16INK4A. Cells were collected at 48 hours post treatment and western blotted for the presence of Rb and Actin. D) CEM, Jurkat, H9, and U937 uninfected cell lines, as well as, J1.1 and U1 latently infected HIV-1 cell lines were assayed for the presence of endogenous levels of p16INK4A, cdk4, and Rb. One hundred micrograms of whole cell extract from each cell line was probed with antibodies against p16INK4A, cdk4, Rb, and Actin using Western blots. E) Uninfected/Infected cell line pairs Jurkat/J1.1 and U937/U1 were treated with GST or GST-p16INK4A (0.5 μg) to test for entry of GST-p16 into the cells. Cells were collected at 48 hours post treatment, washed, lysed, and incubated with Glutathione-Sepharose beads overnight. Beads were washed extensively, and probed for the presence of GST-p16INK4A by western blot against p16INK4A. Arrows indicate the endogenously expressed p16INK4A in the control Jurkat lane as well as the larger, GST-p16INK4A band.