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. 2017 Jul 27;13(7):e1006896. doi: 10.1371/journal.pgen.1006896

Fig 3. miR-1254 represses HO-1 transcription.

Fig 3

(A) Sequence alignment of wild-type/5mt miR-1254 with wild-type/mutant HO-1 3`UTR. (B) Luciferase activity in HEK293 cells transfected with miR-1254 mimics and reporter plasmids containing wt or mt HO-1 3′-UTR normalized to activity in cells transfected with nc. (C and D) HO-1 mRNA (C) and protein (D) levels in A549 cells transfected with the indicated nucleotides (MiR-1254 and its 5`mt) for 48 h were measured by qRT-PCR and immunoblotting respectively. (E) The effect of miR-1254 on HO-1 mRNA stability. A549 and NCI-H1975 cells were transiently transfected with the indicated oligonucleotides, and after 4 hours each culture was treated with 5 μg/ml actinomycin D (ActD) for the indicated times. (F) ChIP analysis of the polⅡ binding to the HO-1 promoter in the A549 cells using antibodies against polⅡ, with IgG as a negative control. Top: Representative images. Bottom: Statistical results. (G) Nucleotides with mutations (underlined and italic) used in all experiments. (H and I) Luciferase activity in HEK293 cells (H) and HO-1 mRNA expression in A549 cells (I), cells were all transfected with indicated oligonuclitides and harvested 48h post-transfection. Data are presented as the mean ± SEM of three independent experiments. *P <0.05, **P and ***P <0.01 vs. nc; #P <0.05, ##P and ###P <0.01 vs miR-1254.