Liver-enriched transcription factors regulate MicroRNA-122 that targets CUTL1 during liver development

Additional Supporting Information may be found in the online version of this article.

Filename	Format	Size	Description
HEP_23818_sm_Suppfig1.tif		12713K	Supplementary Figure 1. The expression of all known LETFs in human (A) and mouse (B) tissues. We search the six families of LETFs in the UCSC Genome Browser by Gene Sorter, and their expression data was downloaded and gathered.
HEP_23818_sm_Suppfig2.tif		2314K	Supplementary Figure 2. BLAT result shows the conservation of primary transcript for miR-122 between various animals. Sequence for the query is the known genomic sequence of woodchuck hcr (primary transcript for miR-122). Sequence information see the reference 22.
HEP_23818_sm_Suppfig3.tif		4194K	Supplementary Figure 3. 5'RACE results show the transcription start sites of human (Huh7 cell) and mouse pri-miR-122. A. Agarose gel electrophoresis shows the PCR products of 5'RACE. B. Alignment of genomic sequence shows the detailed transcription start sites of pri-miR-122. The translation start sites of pri-miR-122 are underlined. In woodchuck, it located at S1 site. In human (Huh7 cell), pri-miR-122 could start at two sites (S2 and S5), whereas it could start at four sites in mouse (S1, S2, S3 and S4). The numbers indicate the frequency of each site in the sequenced clones. 8 and 12 clones were sequenced for human and mouse RACE products respectively.
HEP_23818_sm_Suppfig4.tif		162K	Supplementary Figure 4. qRT-PCR assays show the effect of the miR-122 mimic (122) on the expression of target genes in HepG2 cells. The level of target genes was normalized to GAPDH and then compared to the samples transfected with control mimic (NC)
HEP_23818_sm_Suppfig5.tif		4099K	Supplementary Figure 5. Multiple sequence alignment of the binding site for miR-122 within CUTL1 3'UTR in 43 species. This picture is a result of Blat from the UCSC Genome Browser. The sequence for Blat is the 47-bp fragment of CUTL1 gene that was used for target validation (5'-TGG GTT TTG CAG ACC AGG GTT TGT TTA ATA CAC TCC ATT CTA GGC CA -3', it is also shown in Supplementary Table 3.). The putative binding site for miR-122 seed is shown as green box in the picture.
HEP_23818_sm_Suppfig6.tif		3831K	Supplementary Figure 6. A. Flow cytometry analysis shows the effect of the miR-122 mimic on the cell cycle of HepG2 cells. Cells were transfected with 50 nM miR-122 mimic (122) or the control mimic (NC) and were collected, stained and analyzed 48 h after transfection. B. Cell growth assay shows the effect of the miR-122 mimic on the proliferation of HepG2 cells. Cells were transfected with 50 nM of the mimics. 24 h following transfection, cells were trypsinized and seeded into 6-well-plates at 3000 cells per well and maintained for 10 days. Then, the cells were fixed and stained by crystal violet.
HEP_23818_sm_Suppfig7.tif		680K	Supplementary Figure 7. Western blot assay shows the expression of SRF in developing mouse livers, HCC cell lines as well as the effect of over-expression of miR-122 on SRF protein expression. All the samples used were the same as before.
HEP_23818_sm_Supptable.doc		159K	Supplementary Table 1. Real-time PCR primers for miR-122, U6, LETFs and GAPDH. Some primers are different between mouse (M) and human (H) and the different bases are underlined.
HEP_23818_sm_Suppinfo.doc		711K	Supporting Information.
HEP_23818_sm_Suppreference.doc		27K	Supporting Information.

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