Figure 1.
Effects of miR-122 and miR-34a on FUT8 and ALDOA 3′UTRs in a luciferase reporter assay.
For each experiment, HeLa cells were seeded in 96 plates (six replicates for each condition) and they were then co-transfected with either miR-122 or miR-34a mimics and with empty pMiR-Report, with pMiR-Report containing downstream FUT8 3′UTR (pMiR-Report/FUT8) or ALDOA 3′UTR (pMiR-Report/ALDOA). Luciferase activity was determined after 24 hours post-transfection. Control = AllStar siRNA negative control. Data were expressed as luminescence units (RLU) means ± SD of six replicates in three independent experiments. *p<0.05.
Figure 2.
Effects of miRNA transfection HepG2 cells.
Cells were transfected with either the AllStar siRNA negative control, miR-122 mimic or miR-34a mimic in 12 well plates. After 24 and 48 hours total RNA and proteins were extracted. (A) mRNA levels were determined by qRT-PCR. Data were reported as relative fold expression compared to controls (indicated by the dotted line) and they were means ± SD of seven independent experiments. (B) Total proteins of HepG2 were recovered after 24 and 48 hours post-transfection of AllStar siRNA negative control, miR-122 or miR-34a mimics. After SDS-PAGE separation and blotting, the membranes were probed sequentially using antibodies against FUT8, ALDOA and β-Actin. Band intensities were measured by densitometry and the values obtained for FUT8 and ALDOA were normalized using β-Actin. Data are expressed as normalized levels relative to the cells transfected with the negative control (indicated by the dotted line). Data were obtained from three independent experiments. *p<0.05.
Figure 3.
Effects of miRNA transfection on Fut8 protein expression in rodent hepatocarcinoma cell lines.
mRNA levels were determined by real-time PCR and were normalized to the value obtained for cell transfected with the siRNA negative control. Western blots were performed using anti-FUT8 antibody; after stripping the same membrane was probed with an anti-β-actin antibody. Data are from one representative experiment for each cell line. Mouse Hepa1C1C7 cell line: Fut8 mRNA (A) and protein (C) levels. Rat HTC cell line: Fut8 mRNA (B) and protein (D) levels.
Figure 4.
Lectin blot analysis of culture media from HepG2, Hepa1C1C7 and HTC cells after miRNA mimic transfection.
Secreted proteins recovered from cell culture supernatants were run on a 10% SDS-PAGE gel and proteins were then blotted on a PVDF membrane. The membrane was probed with biotinylated-LCA and revealed with streptavidin-HRP. Parallel samples were also extensively treated with PNGase F in order to remove N-linked glycans and ensure the specificity of the signal. Representative experiments are shown.