Impact of hPMR1 knockdown on albumin mRNA decay. (A) U2OS cells were transduced with lentivirus expressing a scrambled control shRNA (scr; lane 2) and shRNA against hPMR1 (lane 3). Knockdown efficiency was determined by Western blotting with an anti-PXDNL (hPMR1) antibody (upper panel) or antibody to GAPDH (lower panel). (B) Triplicate cultures of the cell lines transduced with the scrambled shRNA (open bars) or hPMR1 shRNA (filled bars) were transfected with albumin and luciferase reporter plasmids 16 h prior to adding DRB to block transcription. Cytoplasmic RNA recovered at the time of DRB addition (t = 0) and 8 h after DRB was analyzed by quantitative RT-PCR, as in Figure 6B. (C) Triplicate cultures of HEK293T cells were transfected twice at 24-h intervals with siRNA targeting hPMR1 or a scrambled control, and knockdown efficiency was determined by as in A. (D) Plasmids expressing albumin and luciferase mRNA were added to the second round of siRNA transfection, followed 16 h later by addition of DRB. Cytoplasmic RNA recovered at the time of DRB addition and 8 h later was assayed as in B. In B and D, the mean value at time 0 for albumin mRNA normalized to luciferase mRNA was arbitrarily set to 1; error bars, SD; **P < .001 by Student's t-test.
