Fig. 5.

A: box-and-whisker plot showing the relative luciferase activity in isolated neonatal cardiac fibroblasts transfected with firefly and Renilla luciferase reporter pEZX-MT01 (control) or pEZX-MT01 [myocardial infarction-associated transcript (MIAT)]. Cells were also cotransfected with pre-miR-29 family (miR-29a, -b, -c; 50 nM) or scramble oligonucleotides (NC). Ratio of firefly to Renilla was determined after 48 h and reported as relative luciferase activity compared with NC, which was independently set as 1. The results were analyzed using unpaired t test, and similar results were obtained from 4 sets of independent experiment. *P < 0.05 and **P < 0.01 vs. NC. B–D: RNA immunoprecipitation with argonaute 2 (Ago2) antibody was conducted to confirm the association between MIAT and miR-29 family using cell lysates from neonatal cardiac fibroblasts following nicotine (10⁻⁹ M) treatment for 96 h. B: representative image from 10% volume of the beads suspension after immunoprecipitation was applied to Western blotting for Ago2 antibody efficiency test. The expression of MIAT (C) and miR-29 family (D) from purified RNA was determined by qRT-PCR. The results are presented as box-and-whisker plot from 6 sets of independent experiments and were analyzed using unpaired t test. *P < 0.05; **P < 0.01, and ***P < 0.001 vs. IgG; #P < 0.05 and ##P < 0.01 vs. Ago2 of the control group. Thin horizontal lines within boxes are medians, cross symbols are means values, and whiskers indicate maximum and minimum values.