Fig 1.

The regulatory roles of NF90 on miR-548k. (A) After transfection of NF90 overexpression or control plasmids into KYSE30 cells, NF90 expression were measured by western blot. (B) After transfection of NF90 overexpression or control plasmids into KYSE30 cells, miR-548k expression were measured by qRT-PCR. (C) After transfection of NF90 specific or control shRNAs into Eca-109 cells, NF90 expression were measured by western blot. (D) After transfection of NF90 specific or control shRNAs into Eca-109 cells, miR-548k expression were measured by qRT-PCR. (E) RIP assay followed by qRT-PCR was performed to detect the specific enrichment of pri-miR-548k with NF90 specific antibody compared with nonspecific IgG. pri-miR-21 was used as negative control. (F) After transfection of NF90 overexpression or control plasmids into KYSE30 cells, the stability of pri-miR-548k transcript over time was evaluated by qRT-PCR relative to time 0 after blocking new RNA synthesis with α-amanitin and normalized to 18S rRNA (transcribed by RNA polymerase I and not influenced by α-amanitin). (G) After transfection of NF90 specific or control shRNAs into Eca-109 cells, the stability of pri-miR-548k transcript over time was evaluated by qRT-PCR relative to time 0 after blocking new RNA synthesis with α-amanitin and normalized to 18S rRNA. (H) After transfection of NF90 overexpression or control plasmids into KYSE30 cells, pri-miR-548k expression was measured by qRT-PCR. (I) After transfection of NF90 specific or control shRNAs into Eca-109 cells, pri-miR-548k expression was measured by qRT-PCR. Results are presented as mean ± S.D. (n = 3). **P < 0.01, ***P < 0.001, ns, not significant, by Student's t-test (A, B, E, F and H) or one-way ANOVA followed by Dunnett's multiple comparison test (C, D, G and I).