Supplmentary Figure Legends
SupplementaryFigure S1. Quantitative RT-PCR Validation of miR-19b, -100, -125b and -205 in Esophageal Cultured Cells. Microarray and qRT-PCR results are shown side-by-side in normal esophageal epithelial cells (HEEpiC), BE-derived cell lines (QhTRT, ChTRT and GiHTRT), and an EAC-derived cell line (OE-33). The value of HEEpiC was set at 1. qRT-PCR was performed in triplicate, and error bars indicate standard deviations. Results of qRT-PCRs correlated closely with those of microarray assays.
SupplementaryFigure S2. Quantitative RT-PCR of miR-100, -125b and -205 in Esophageal Tissues.22 NE, 24 BE, and 22 EAC specimens were analyzed. The average value of NE tissues was set at 1. Horizontal bars indicate average levels. MiR-100, -125b and -205 expression levels were significantly downregulated in both BE and EAC relative to NE, with the sole exception of miR-125b in BE. These results correlated closely with data obtained from cultured cells. NE: normal esophageal epithelial tissues; BE: Barrett’s esophagus tissues; EAC: esophageal adenocarcinoma tissues; NS: not significant.
Supplementary Figure S3. OE-33 Natural Growth Curve in Nude Mice. 2 x 106 OE-33 untreated cells were implanted subcutaneously into the flanks of nude mice (N=4 each). An error bar indicates standard deviations.
SupplementaryFigure S4. MiR-25, -93 and -106b Expression Following miR Mimic Transfection.MiR-25, -93 and -106b expression levels were measured by qRT-PCR at 4 and 6 days following miR mimic transfection (60nM) in WI-38 cells. “3 miRs-MM (mimic)” denotes a cocktail containing 20nM of each of miRs -25, -93, and -106b (total miR concentration, 60nM). Data were normalized to RNU6B, and the value obtained withthe nonspecific control-miR mimic (NSC-MM) was set at 1. MiR mimic transfection was detected as upregulation of these miRs at days 4 and 6. MiR-25, -93 and -106b expression levels were higher at day 4 than at day 6. MiR expression after individual miR-MM transfections (60nM) was higher than after transfecting with the cocktail of 3 miRs-MM (20nM each). NT: untreated cells.
SupplementaryFigure S5. Western Blotting for p21 and Bim Using in vivoTumor. Western blotting assays for p21 (A) and Bim (B) expression byin vivo tumors at day 11 (i.e., 12 days following miR transfection). These results corroborate those of in vitro experiments regarding the effect of miRs -93 and -106b on p21 and of miR-25 on Bim (Figures 5B and 6B).
SupplementaryFigure S6. Luciferase assays for p21 and Bim 3’UTR using universal negative control plasmids. Plasmid clones containing reverse-oriented inserts (pGL4.13-revP21UTR for A and B, and pGL4.13-revBimUTR for C and D) were used as universal negative controls. (A and B) Luciferase activity was significantly decreased by miR-93-MM and miR-106b-MM in both ChTRT (A) and GihTRT (B) cells, but not by miR-25-MM. (C and D) Luciferase activity was significantly decreased by miR-25-MM in QhTRT cells (C) and significantly increased by miR-25-INH in OE-33 cells (D), but not by miR-93-MM and miR-106b-MM.
SupplementaryFigure S7. Ki-67 staining of OE-33 cells transfected with miR inhibitors. Ki-67 staining of OE-33 cells after 48-hour transfection with NSC-INH (A), miR-25-INH (B), miR-93-INH (C) and miR-106b-INH (D) is shown. The number of Ki-67 positive cells is diminished in miR-25-INH-, miR-93-INH-, and miR-106b-INH transfected cells, relative tothe number in NSC-INH transfected cells.