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

HEP_22919_sm_SupportingDocument.doc42KSupporting Materials and Methods
HEP_22919_sm_SupportingTables.doc94KSupplementary Table 1. The Characteristics of Patients and Expression Status of miR-195 and Its Target Genes
HEP_22919_sm_SupportingFigure1.tif264KSupporting Figure 1. Specific detection of miR-195 by Northern blot. (A) Optimal hybridization temperature for specific detection of miR-195. Five pmols of synthetic RNA duplexes corresponding to miR-195 (lane 1) and miR-16 (lane 2) were separated on 15% denaturing polyacrylamide gel. Hybridizations were performed at indicated temperatures. The probe sequence was complementary to mature miR-195. (B) Sequence alignment of mature miRNA sequences of the miR-15/16/195 family members. Shading letters indicate the nucleotides same as mature miR-195.
HEP_22919_sm_SupportingFigure2.tif902KSupporting Figure 2. Expression level of miR-195 in different cell lines and normal human liver tissue. The same membrane was hybridized sequentially with miR-195 and U6 probe, as indicated on the left. HCC, hepatocellular carcinoma cell lines; U6, control for RNA loading.
HEP_22919_sm_SupportingFigure3.tif190KSupporting Figure 3. miR-195 expression reduces the number of viable cells. Cells were first transfected with NC or miR-195 duplex in a 24-well plate for 24 h, and then re-plated into 96-well plate at about 10% confluence, followed by Alamar Blue assay at every 24 h for 5 days, indicated as Day 1 to 5. *, P < 0.05; ***, P < 0.001, compared with NC-transfectants.
HEP_22919_sm_SupportingFigure4.tif460KSupporting Figure 4. miR-195 suppresses anchorage-independent cell growth. Representative pictures (A) and quantitative plots (B) are shown. Colonies larger than 150 μm in diameter were counted under 30 separate microscopic fields at 150 × magnification. ***, P < 0.001, compared with NC-transfectants.
HEP_22919_sm_SupportingFigure5.tif210KSupporting Figure 5. Ectopic expression of miR-195 induces accumulation of G1-cells in hepatocellular carcinoma (HCC), colorectal carcinoma (CRC) and osteosarcoma cells. For measurement of apoptotic cells, nuclear debris detected as sub-G1 peak were included. The percentage of G1 population is indicated within each histogram.
HEP_22919_sm_SupportingFigure6.tif795KSupporting Figure 6. Rb-E2F signaling is involved in miR-195-regulated G1/S transition in U-2OS cells. (A) Overexpression of miR-195 and knockdown of either cyclin D1, CDK6 or E2F3 increase the proportion of G1-cells. (B-C) miR-195 expression delays S-phase entry upon serum stimulation as assessed by FASC analysis (B) and BrdU incorporation assay (C). **, P < 0.01; ***, P < 0.001, compared with NC-transfectants. (D) Suppressed expression of endogenous cyclin D1, CDK6 and E2F3 by miR-195 or siRNA. Forty-eight hours after transfection with indicated RNA duplex, endogenous mRNA and protein levels of cyclin D1, CDK6 and E2F3 were analyzed by RT-PCR and Western Blot, respectively. (E) Phosphorylation of Rb protein is inhibited by miR-195. pRb, total Rb protein; ppRb, phosphorylated pRb. (F) Transactivation of E2F3 target genes is impeded by miR-195. The time point when serum was re-added was set as 0 h. Gene expression was examined by RT-PCR. β-actin or hPRT was used as internal control.
HEP_22919_sm_SupportingFigure7.tif174KSupporting Figure 7. Effect of miR-16 and miR-497 overexpression on the G1-population. HCT-116 cells were transfected with indicated miRNA duplex. Nocodazole (40 ng/ml) was added 32 h after transfection and cells were cultured for additional 16 h, followed by FASC analysis. ***, P < 0.001, compared with NC-transfectants.
HEP_22919_sm_SupportingFigure8.tif148KSupporting Figure 8. Putative miR-195 binding sites in the 3'UTR of respective genes. Mutation was generated in the complementary site (underlined) for the seed region of miR-195, as indicated.
HEP_22919_sm_SupportingFigure9.tif443KSupporting Figure 9. Examination of c-Myc expression by RT-PCR and Western blotting. (A) Expression of endogenous c-Myc in U-2OS, MHCC-97L and HCT-116 cells. (B) Knockdown of c-Myc expression in HCT-116 cells by 5 nM of siMyc. β-actin was used as internal control.
HEP_22919_sm_SupportingFigure10.tif120KSupporting Figure 10. Expression level of miR-195 and miR-16 after c-Myc knockdown. Levels of miR-195 and miR-16 were analyzed by real-time RT-PCR assay (PN 4373105 and PN 4373121, ABI, Foster City, CA) at indicated time points after transfection with siMyc or NC duplex in HCT-116 cells. RNU49 was used as internal control (PN 4373376, ABI). Fold change of expression indicates miRNA expression in si-Myc-transfectants relative to that of the matched NC-transfectants.
HEP_22919_sm_SupportingFigure11.tif458KSupporting Figure 11. Effect of miR-195, miR-16 or miR-497 overexpression on endogenous cyclin D1 expression of HCT-116 cells. Forty-eight hours after transfection with indicated RNA duplex, endogenous protein levels of cyclin D1 was assayed by Western Blot. β-actin was used as internal control.

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