Figure 1

Validation of PATZ1 as a target of miR-29b.

(a) predicted miR-29b/ PATZ1 alignment, according to microRNA.org web system. mirSVR (cutoff 0.1 or lower) and PhastCons (cutoff 0.57 or higher) are downregulation and conservation scores, respectively. (b) Western blot using anti-PATZ1 on HEK293 total cellular extracts collected 72 h after transfection with increasing amount (50–100 nM) of synthetic miR-29b precursor or scramble (100 nM) oligonucleotide. Vinculin was used for normalization. Relative expression levels, 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. (c) qRT-PCR on total RNA from HEK293 cells previously transfected with 100 nM synthetic miR-29b precursor or scramble oligonucleotide. PATZ1 mRNA levels were normalized for endogenous G6PD levels. The mean ± SE of four independent experiments performed in duplicate is reported. (d) Luciferase assay on HEK293 cells co-transfected with the Luc-PATZ1-3′UTR and pCMV renilla reporter vectors along with 100 nM synthetic miR-29b precursor or scramble oligonucleotide. Relative firefly luciferase activity levels were normalized for renilla luciferase activity and analysed relatively to scramble-transfected cells, which were set to 1. The mean ± SE of four independent experiments performed in duplicate is reported. ****P < 0,0001.