Fig 2.
vGPCR increases the half-life of endogenous ARE-containing mRNAs. (A to E) HUVE cells were transduced with recombinant retroviruses expressing vGPCR, a signaling inactive mutant of vGPCR (vGPCRR143A), or the empty vector control virus. After 2 days of selection with puromycin and 1 day of recovery, cell monolayers were examined by light microscopy (scale bar in panel A = 200 μm). Transduced cell monolayers were also analyzed as follows: cells were seeded on coverslips overnight and then fixed and immunostained using anti-vGPCR antibody (scale bar = 10 μm, thick arrows indicate high-expressing cells while thin arrows indicate low-expressing cells) (B); cells were lysed in protein sample buffer, and 25 μg of protein was analyzed by SDS-PAGE and immunoblotting with anti-Cox-2 antibody at 1:500, anti-vGPCR antibody at 1:2,000, or anti-β-actin antibody at 1:5,000 (C); ELISA analysis of cell-free supernatants was done for the presence of human IL-8 and IL-6, which are encoded by ARE-containing RNAs (D); cells were harvested for total RNA isolation, and the steady-state levels of the Cox-2 and IL-6 transcripts were determined by RT-qPCR (E). Error bars represent standard deviations (n = 3 experiments). (F) HUVE cells transduced as described in the legends to panels A to E were either treated with actinomycin D, which stops de novo transcription, for 1, 2, or 3 h, or not treated and harvested for total RNA. Each sample was analyzed by RT-qPCR to determine the decay rates of the Cox-2 transcript. Values are presented as fractions of the transcript levels at the time of actinomycin D addition, and the error bars represent standard deviations (n = 3 experiments). (G) Log2 values of the data from panel E were plotted versus time independently for all three replicates and fitted with the linear regression curves. From the linear regression analyses, the slope value m was obtained. The inverted negative values of each m value are the calculated half-life values shown on the plot (t1/2 = −1/m).