Figure 2

miR-29b targeting of PATZ1 in rat thyroid cells.

(a) Western blot using anti-PATZ1 on PC Cl3 total extracts previously transfected with synthetic miR-29b precursor or scramble oligonucleotide. Three major specific bands were observed (arrows). Vinculin was used for normalization. Densitometric analysis by Image J software was applied on the gel: Relative expression levels of PATZ1, compared to scramble-transfected control and normalized with respect to vinculin, are indicated on the bottom. Black lines delineate the boundary between not contiguous lanes of the same gel. (b) qRT-PCR on total RNA from PC Cl3 and FRTL-5 cells previously transfected with synthetic miR-29b precursor or scramble oligonucleotide. PATZ1 mRNA levels were normalized for endogenous G6PD levels. The mean ± SE of three independent experiments performed in duplicate for each cell line is reported. *P < 0.05 compared with scramble transfected control. (c) qRT-PCR on total RNA from FRTL-5-Ras inducible cells treated with Tamoxifen at the indicated times and FRTL-5 clones V27 and V29 stably expressing the Ha-Ras V12 oncogene. miR-29b and PATZ1 expression levels were normalized for endogenous U6 and G6PD levels, respectively. The mean ± SE of one experiment performed in triplicate and three independent experiment performed in duplicate is reported for miR-29b and PATZ1, respectively. **P < 0.01; ***P < 0.001; ****P < 0.0001 compared with mock-treated or mock-transfected cells for FRTL5-Ras inducible cells and stable clones, respectively. (d) Western blot using anti-PATZ1 on total extracts from cell as in C. The three specific bands corresponding to different PATZ1 isoforms are indicated by arrows. Normalized expression levels of PATZ1, as assessed by densitometric analysis on the upper PATZ1-specific band with respect to vinculin expression, are indicated on the bottom.