FIG. 3.

Analysis of activity of the generated TTF-1/Si constructs and establishment and characterization of FRTL-5 cell lines with TTF-1 depletion by RNAi. (A) HEK-293 cells were cotransfected with CMV-TTF-1 plasmid (4 μg) and one silencing construct (2 μg each of TTF-Si1, TTF-Si2, or control vector[Ctr] as indicated). Nuclear extracts were immunoblotted with antibody against TTF-1. (B) FRTL-5 cells were transfected with TTF-Si2 (5 μg) or Ctr-Si and pPURO (0.5 μg) and grown with 1 μg/ml puromycin for 2 weeks. Nuclear extracts from clones 11 and 7 were analyzed for TTF-1 expression by immunoblotting. The membrane was subsequently stripped and reprobed with anti-YY-1-specific antibodies for loading control. Note that, as previously described (14), TTF-1 appears as two bands in thyroidal cells (B), whereas only the higher-molecular-weight band is visible in transfected nonthyroid cells (A). (C) FRTL-5 clones stably expressing N188K or the TTF-1 Si2 constructs were transiently transfected with C5-CAT and CMV-Luc as internal control. The C5-CAT/CMV-Luc ratio in control cells (pcDNA) is arbitrarily reported as 100%. Values ±SD are the mean of at least three experiments.