Supplemental figure1 Parkin is involved in transcriptional regulation of follistatin. (a) Human

hepatoma-derived cell line, Hep3B, was transiently transfected with an expression plasmid encoding parkin or control vector (CTR). Cell lysates were prepared 24 h after transfection and immunoblotting was performed using antibodies specific for human anti-parkin (upper panel), anti-follistatin (Fst) (middle panel) or anti-α-tubulin (lower panel).(b)Huh-7 or Hep3B cells were transiently transfected with expression plasmid encoding wild-type parkin, a mutant form of parkin lacking exons 3-4 (mut), or control vectors (CTR). Then, total RNA was extracted from the cells and subjected to semi-quantitative (upper panels) or quantitative real-time RT-PCR analyses (lower graphs) for follistatin (Fst). The amounts of follistatin mRNA were normalized to an endogenous reference gene (18S-rRNA) and values shown in the graphs were normalized relative to the control cells (mean  SD; n=3). *P value of < 0.05 as determined by Student’s t-test.(c)Hep3B cells were transfected with 0, 0.5, 1.0, or 2.0 μg of expression plasmids for wild-type human parkin, normalizing total DNA to 2.0 μg using empty pcDNA3. After 24 h, total RNA was extracted and semiquantitative RT-PCR was performed using primers specific for parkin (upper panel), follistatin (Fst) (middle panel) and -actin (lower panel). (d) Luciferase assay for follistatin gene regulation by parkin. Schematic diagram of reporter plasmids comprising the 5’-end sequences of follistatin gene relative to the start of exon 1 are shown (upper panel). NIH3T3cells were trasnfected with the reporter plasmids encoding the promoter region of follistatin gene together with an expression plasmid encoding parkinor control vector (pcDNA3). A whole cell lysates were prepared and assayed for luciferase activity. The data represent means of the relative luciferase activities in three independent experiments. *P value of < 0.05 as determined by Student’s t-test.

Supplemental figure 2. Parkin and follistatin(Fst) mRNA expression in human HCC and its surrounding nontumorous liver tissues. (a, b) Comparison of parkin and follistatin(Fst) mRNA expression in HCC and its surrounding noncancerous liver tissues. The representative results of RT-PCR analyses on the expression levels of parkin and follistatintranscripts are shown. (a) Case#1-#5 that revealed the lack of parkin expression in tumor tissues had the high expression of follistatin transcripts. (b) Case#6 and #7 that had the comparable levels of parkin expression in tumor to those in the surrounding nontumorousliver tissues lacked the upregulation of follistatin.

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