1. Volume 21, Issue 6, Pages 1204-1211 (June 2013)
Chemically Modified Synthetic microRNA-205 Inhibits the Growth of Melanoma Cells In Vitro and In Vivo 
Shunsuke Noguchi, Junya Iwasaki, Minami Kumazaki, Takashi Mori, Kohji Maruo, Hiroki Sakai, Nami Yamada, Kazuyuki Shimada, Tomoki Naoe, Yukio Kitade, Yukihiro Akao 
Molecular Therapy 
Volume 21, Issue 6, Pages (June 2013)
DOI: /mt
Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

2. Figure 1
Sequences of the variants of miR-205 with or without BP used in this study. The sequence of Pre-miR-205 is same as that of the wild-type miR-205 (miR-205/S1).
Molecular Therapy  , DOI: ( /mt )
Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

3. Figure 2
Number of viable A2058 and Mewo cells transfected with control miRNA, Pre-miR-205, or each synthetic miR-205BP (10 nmol/l). Cell counts were performed at 96 (A2058) or 120 (Mewo) hours after the transfection. *P < 0.05, **P < P values were determined for differences between the cells transfected with control miRNA and those transfected with Pre-miR-205 or each miR-205BP tested. Means + SD indicated by error bars are shown.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

4. Figure 3
The effects of transfections with Pre-miR-205 or miR-205BP/S3 on the protein expression levels of target genes of miR-205 and the related genes. (a) Protein expression levels of validated miR-205 target genes, E2F1 and VEGF, downstream of E2F1, BCL2, and PARP-1 in A2058 and Mewo cells. The numerical values under the panels indicate the densitometric value (/β-actin). Extraction of protein was performed at 96 (A2058) or 120 (Mewo) hours after the transfection. (b) Number of apoptotic cells, which were identified by fragmentation or aggregation of nuclei under Hoechst33342 staining, in A2058 cells transfected with each miRNA. *P < P values were determined for differences between the cells transfected with each miRNA. Hoechst33342 staining was performed at 96 hours after the transfection. Means + SD indicated by error bars are shown.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

5. Figure 4
Luciferase activities after cotransfection with control miRNA, Pre-miR-205, or miR-205BP/S3 and each of the sensor vectors with the wild or mutated 3′-UTR of E2F1. The upper panel shows the regions of the 3′-UTR of human E2F1 mRNA complementary to mature miR-205. The red box indicates the predicted binding sites for miR-205. *P < P values were determined for differences between the bracketed samples. Data are expressed as the means + SD indicated by the error bars.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

6. Figure 5
The effect of E2F1 knockdown in A2058 cells. (a) Comparison of the number of viable cells between A2058 cells transfected with control miRNA and those treated with siR-E2F1. (b) Protein expression levels of E2F1 and the predicted signaling molecule downstream of E2F1, BCL2, and PARP-1 in A2058 cells transfected with control miRNA or siR-E2F1. (c) Number of apoptotic cells, which were identified by fragmentation or aggregation of nuclei by Hoechst33342 staining, among A2058 cells transfected with control miRNA or siR-E2F1. The assay was performed at 96 hours after the transfection. *P < Means + SD indicated by error bars are given.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

7. Figure 6
Decay of the various variants of miR-205 in (a) fetal bovine serum or (b) in mice xenografted tumor tissues, evaluated by performing real-time reverse transcriptase PCR using the TaqMan miRNA assay. (a) The % of miR-205 remaining is expressed for each sample indicated in the figure. (b) The absolute expression level of miR-205 based on the standard curve method is expressed. *Significant difference between the cells transfected with Pre-miR-205-5p and *those with miR-205BP/S3, §those with miR-205/S3 and those with miR-205BP/S3, and ¶those with miR-205BP/S1 and those with miR-205/S1. *,§,¶P < Means + SD indicated by error bars are given.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

8. Figure 7
Tumor-suppressive effects of miR-205/S3 in mice bearing xenografted A2058 cells. (a) Time course of tumor size in mice injected with control miRNA, Pre-miR-205 or miR-205BP/S3. P values were determined for differences *between the tumors injected with control miRNA and those injected with miR-205/S3, ¶between the tumors injected with Pre-miR-205 and those injected with miR-205BP/S3 or §between the tumors injected control miRNA and those injected with Pre-miR-205. *,¶,§P < (b) Comparison of weight of tumors removed from mice at the day of sacrifice, with miRNAs injected into the tumors indicated on the abscissa. *Significant difference between bracketed samples (*P < 0.05). Means + SD indicated by error bars are given. (c) Expression levels of E2F1, VEGF, and BCL2 in the tumors injected with each miRNA are shown in the upper panel. The graphs in the lower panel show the relative expression level of E2F1, VEGF, or BCL2 normalized to β-actin. Means ± SD indicated by error bars are given. (d) A flow diagram on the miR-205 on its target genes and the related pathways. S, sacrifice; T, transplantation; triangles, intratumoral injection.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions