The mixture was then transferred to a streptavi din different coated plate. The bound NF B transcription factor subunits p50 and p65 were detected with specific primary antibodies. A horseradish peroxidase conjugated second ary antibody was then used for chemiluminescent detec tion. The relative light unit values were measured using a LUMIstar Omega microplate reader. Statistical Analysis Differences between groups were assessed by using one way ANOVA with the SPSS 13. 0 program, with a probalility value of P 0. 05 considered indic ative of statistically significance. Results MTT Cell Viability Studies To exclude the possibility that changes in the barrier func tion resulted from cell death and the subsequent forma tion of holes in the monolayer, we tested the cytotoxic effects of 100 nM Tat on D407 cells.

As shown in Figure 1, the average absorbance at 490 nm did not differ signifi cantly between the control and treatment groups, indicat ing that the exposing cells to 100 nM Tat for 24 72 hours did not decrease cell viability relative to controls. HIV 1 Tat Induces destruction of barrier function in RPE The TER appeared to be somewhat affected by the serum, so we reduce the serum concentration of the medium to 1% from day 3 when cells reached confluence, and meas ured the TER every other day. The TER of D407 cells grad ually increased on the subsequent days, peaking at day 8 and then remaining stable for 1 week. Mennel suggested that obtaining stable values on 2 subse quent days indicated the formation of a tightly coupled cell monolayer, and hence we decided to begin treating the cells with 100 nM Tat from day 10.

The TER of D407 cells was measured at 1, 2, 3, 12, 24, 48, and 72 hours after treatment with 100 nM Tat. A reduc tion in the TER was first evident after 3 hours of treatment. Continuous culturing of cells for longer peri ods further reduced the TER, with a maximum effect after 24 hours of treatment that was maintained to 72 hours. The TER of control groups remained unchanged throughout the experiment. The permeability to sodium fluorescein, which has a low molecular weight, is regarded as a reliable marker of para cellular permeation. The permeability values of cells as measured at 20, 40, and 60 min after treatment with 100 nM Tat for 24 hours were all significantly higher than those of cells in the standard medium and the Hi Tat con tained medium, indicating that treating D407 cells with 100 nM Tat for 24 hours induced a loss of junctional integrity.

HIV Dacomitinib 1 Tat Induces Genes and Proteins Expression of TJs in RPE The real time quantitative reverse transcriptase polymer ase chain reaction demonstrated that occludin and clau din 1 to 4 were expressed in D407 cells, whereas there was no expression of claudin 5, similar to those from studies on claudins in another RPE cell line ARPE19.