Figure 6From: Triptolide inhibits human immunodeficiency virus type 1 replication by promoting proteasomal degradation of Tat proteinTriptolide reduces Tat protein stability by enhancing proteasomal degradation of Tat. (A) The effect of triptolide on Tat mRNA levels. HeLa cells were transfected with pEF1-Tat in the presence of increasing concentrations of triptolide. Cells were harvested 48 h post-transfection. The steady-state mRNA levels of Tat and GAPDH were examined using semi-quantitative reverse transcription PCR analysis. RT, reverse transcriptase. (B) The effect of triptolide on Tat protein degradation. HeLa cells were transfected with pEF1-FLAG-Tat in the presence of 5 nM triptolide or vehicle (DMSO). At 28 h post-transfection, cells were treated with 20 μg/mL of CHX to arrest protein synthesis. At 0, 1, 2, 3 h post-treatment, cells were harvested to determine protein expression levels using Western blot analysis. (C) The band intensities of FLAG-Tat in panel B were measured, and their natural log values were plotted as a function of time. Results were presented as the mean plus the standard deviations (n = 3). (D) Proteasome inhibition restores Tat protein expression. HeLa cells were transfected with pEF1-FLAG-Tat in the presence or absence of 5 nM triptolide. At 48 h post-transfection, cells were treated with 20 μM of MG132, and cultured for an additional 3 h before harvest. Protein expression levels were determined by Western blot analysis.Back to article page