Fig. 3
miR-380-5p directly targets the ORF of TEP1 and the 3′-UTR of TSPYL5. a Schematic representation of the interaction between miR-380-5p and its predicted TEP1 binding sites. TEP1 sequences used to design target protectors are also shown. Numbers in brackets indicate the nucleotide position within TEP1 mRNA (NM_007110.4). b Representative RT-PCR showing miR-380-5p target site enrichment. Input: total RNA in cell lysates, AGO2- and IgG-IP: RNA isolated from AGO2- and IgG-immunoprecipitated samples. The graph on the bottom shows TEP1 mRNA enrichment quantification reported as fold-enrichment (mean values ± s.d.) in the indicated immunoprecipitated samples with respect to IgG-IP from NT cells; **P < 0.01. c Representative immunobloting showing TEP1 amounts in preNeg- and miR-380-5p-transfected cells ± target protectors. Cropped images of selected proteins are shown. The graph on the bottom shows the quantification of TEP1 protein (relative amounts with respect to preNeg-transfected cells; mean values ± s.d.); **P < 0.01 vs. miR-380-5p-transfected cells. d Schematic representation of the interaction between miR-380-5p and its predicted binding site within TSPYL5 3′-UTR (NM_033512.2). The mutated sequence (del43-54) bearing a deletion corresponding to miR-380-5p seed and the sequence within TSPYL5 3′UTR used to design the target protector are also shown. (e) Representative immunoblotting showing TSPYL5 amounts in miR-380-5p expressing STO cells. Cropped images of selected proteins are shown. f Time-course assessment of luciferase activity in cells transfected with the wild-type or mutated (del43-54) reporter constructs in the presence of miR-380-5p (percentage of luciferase activity inhibition in miR-380-5p vs. preNeg-transfected cells; mean values ± s.d.); **P < 0.01 TSPYL5 3’UTRwt vs. TSPYL5 3’UTR del43-54. g Assessment of luciferase activity as a function of the different combinations of transfected oligomers. Data have been reported as Gluc/SEAP activity ratio (mean values ± s.d.); *P < 0.05; **P < 0.01 vs. pEX-MT05-transfected cells. Numbers under the X-axis indicate the percent reduction (−) or increase (+) of luciferase activity vs. pEX-MT05-expressing cells. Asterisk in bracket indicates the level of statistical significance (P < 0.05) between LNA380/miR-380-5p and LNANeg/miR-380-5p samples