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HEP_23380_sm_suppfig1.tif1142KSupplementary Fig. 1. Sequences of mature miR-29a/b/c and their putative binding sites. (A) Predicted miR-29b-binding sequences in the 3′-UTR of Bcl-2 and Mcl-1 mRNAs. Mutation was generated in the complementary site for the seed region of miR-29 family members, as indicated. (B) Sequence alignment of the miR-29 family members.
HEP_23380_sm_suppfig2.tif113KSupplementary Fig. 2. Analysis of miR-29 expression. (A) Optimal hybridization temperature for specific detection of miR-29b and miR-29a/c by Northern blot. Five pmol of synthetic RNA duplexes corresponding to miR-29a (lane 1), miR-29b (lane 2) and miR-29c (lane 3) were separated on a 15% denaturing polyacrylamide gel. Hybridizations were performed at 58°C. The same membrane was hybridized sequentially with probes for miR-29a, miR-29b and miR-29c, as indicated on the left. (B) Expression analysis of mature miR-29 in HEK 293T and four human HCC cell lines by Northern blot. The same membrane was hybridized sequentially with miR-29b and U6 probe at 42°C. Hybridization with miR-29b probe revealed a single band that migrated between xylene cyanol FF and bromophenol blue, the loading dyes which co-migrates with ∼30 bp and ∼10bp RNA fragments in 15% denaturing polyacrylamide gel, respectively. U6, internal control for RNA loading. Normal liver, RNA from normal liver tissue. (C) Real-time quantitative RT-PCR analysis of mature miR-29b expression in 17 paired HCC (T) and adjacent non-tumor liver tissues (N). miR-29b level was normalized to U6 expression in each sample. The median values of miR-29b in each group are indicated by a solid horizontal line. The difference between two groups was analyzed by paired Student t test.
HEP_23380_sm_suppfig3.tif1809KSupplementary Fig. 3. miR-29 sensitizes HCC cells to serum deprivation and hypoxia-induced apoptosis. (A) Analysis of cell viability by Alamar Blue. HepG2 cells were first transfected with NC or miR-29a/b/c duplex in a 24-well plate for 24 h, and then replated into 96-well plate at about 20% confluence. Twenty-four hours after splitting, cells were deprived of serum and cultured in 1% O2. Cell viability was evaluated by Alamar Blue assay at the indicated time points. (B–C) Analysis of apoptosis in HepG2 cells by TUNEL staining. Images were captured at 100× magnification. Cells without transfection or transfected with NC or miR-29a/b/c were deprived of serum and cultured in 1% O2 for 24 h, and then subjected to TUNEL staining followed by counterstaining with DAPI. Representative photographs (B) and the apoptotic rates (C) are shown. (D) Effect of anti-miR-29 on exogenous miR-29-promoted apoptosis. HepG2 cells were transfected with indicated RNA dulplex for 24 h, followed by culture in serum-deprived medium and 1% O2 for 72 h before DAPI staining. ***, P < 0.001, comparison between two groups as indicated.
HEP_23380_sm_suppfig4.tif428KSupplementary Fig. 4. miR-29 sensitizes HCC cells to doxorubicin-induced apoptosis. (A) miR-29 sensitizes HepG2 cells to doxorubicin-induced apoptosis in a dose-dependent manner. Cells without transfection or transfected with NC or miR-29a/b/c were treated with indicated concentration of doxorubicin for 48 h before DAPI staining. (B) miR-29 sensitizes QGY-7703 cells to doxorubicin-induced apoptosis. Cells without transfection or transfected with NC or miR-29a/b/c were treated with doxorubicin (0.2 μg /ml) for 72 h before DAPI staining. ***, P < 0.001, compared with NC-transfectants.
HEP_23380_sm_suppfig5.tif88KSupplementary Fig. 5. Enhanced expression of miR-29 decreases the level of endogenous Bcl-2 and Mcl-1 under conditions of serum deprivation and hypoxia. HepG2 cells without transfection or transfected with indicated RNA duplex were deprived of serum and cultured in 1% O2 for 72 h before Western blot analysis. The value under each lane indicates the relative expression level of the putative target gene, which is represented by the intensity ratio between Bcl-2 or Mcl-1and β-actin bands in each lane. β-actin, internal control.
HEP_23380_sm_suppfig6.tif42KSupplementary Fig. 6. Analysis of miR-29b level in miR-29b-transfected HepG2 cells. Cells were first transfected with NC or miR-29b duplex (all pyrimidine nucleotides in the NC or miR-29b duplex were substituted by their 2'-O-methyl analogs) and then maintained in 24-well plate at 37°C with 5% CO2. At every indicated time point, cells were trypsinized and split 1/3 into 24-well plate for sustained culture, and the rest 2/3 cells were applied to RNA isolation and expression analysis of mature miR-29b. The miR-29b level was evaluated by real-time quantitative RT-PCR and normalized by the expression of U6.
HEP_23380_sm_supptabs.doc99KSupplementary Tables
HEP_23380_sm_supptext.doc47KSupplementary Materials and Methods

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