Titles and Legends to Supplementary Figures and Tables

Titles and legends to Supplementary Figures and Tables

Supplementary Figure1

A) Cross resistance of Panc1-P and Panc1-GRs (GR1, GR2, GR3, and GR4) to 5-FU was shown by MTT assay. Data represent mean ± SD of more than three experiments; **, p < 0.01.

B) The representative data of flow cytometry showed CSC-like cell population of Panc1-GR. The percentages of the CSC-like cell population were shown. This assay was performed three times. Data represent mean ± SD of more than three experiments.

C) The proliferation ratios of spheres in the Panc1-P-OE and Panc1-P-C cells wereshown. Data represent mean ± SD of more than three experiments; *, p < 0.05.

D) Growth-inhibitory effects of 5-FU on Panc1-P and Panc1-P (Sp) cells were assessed by MTT assay. Data represent mean ± SD of more than three experiments; **, p < 0.01.

Supplementary Figure2.Gene set enrichment analysis (GSEA) of Panc1-P (Sp) and Panc1-P cells.

A) Other stemness-related gene sets that were enriched in those cells were shown.

B) The more detailed information of GSEA was described.

Supplementary Figure3. The transition of miR-1246 expression after transfection in Panc1 cells.

Real time qRT-PCR showed the transition of miR-1246 expression in Panc1-P cells transfected with pre-miR-1246 (A), Panc1-GR cells transfected with anti-miR-1246 (B), and miR-1246 over-expressing Panc1 cells which continuously expressed high levels of miR-1246 (C).

Supplementary Figure4.Association of miR-1246 expression with the resistance to GEM and sphere-forming ability in MiaPaCa2 cells.

Real time qRT-PCR showed the expression level of miR-1246 in Panc1 and MiaPaCa2 cells (A), and MiaPaCa2 cells which were transfected with pre-miR-1246 (MiaPaCa2-OE) and MiaPaCa2 cells which were transfected with negative control (MiaPaCa2-C) (C). Data represent mean ± SD of more than three experiments; **, p < 0.01.

B) The representative data of flow cytometry showed the CSC-like cell population of MiaPaCa2. The percentages of the CSC-like cell populations were shown. This assay was performed three times. Data represent mean ± SD of more than three experiments.

D) Sphere formation assay was performed in MiaPaCa2-C and MiaPaCa2-OE cells. The representative image of spheres (left) and the number of spheres (right) were shown. Bar = 100 μm. Data are mean ± SD of three experiments; *, p < 0.05.

E) MTT assay demonstrated relative cell survival ratio of MiaPaCa2-C and MiaPaCa2-OE cells to GEM respectively. Data represent mean ± SD of more than three experiments; **, p < 0.01.

G) The representative data of Annexin V assay showed the distribution of the early and late apoptotic cells in MiaPaCa2-C and MiaPaCa2-OE after GEM exposure for 48 hours.

H) The percentages of early and late apoptotic cells in MiaPaCa2-C and MiaPaCa2-OE after GEM exposure for 48 hours. This assay was performed three times. Data represent mean ± SD of three experiments; **, p < 0.01.

Supplementary Figure5. The expression level of miR-1246 and CCNG2 in 24 clinical patients.

A) LCM was performed to collect cancer sections from those 24 specimens. Real time qRT-PCR demonstrated the expression level of miR-1246 in each sample. Relative quantification of miRNA expression was calculated by the comparative CT method (2-ΔCT). The mean value of these samples was calculated and divided into two groups with high or low expression of miR-1246.

B-C) Immunohistochemical staining for CCNG2 in the same 24 pancreatic cancer patients was performed. Normal pancreatic ductal cells (B) and acinar cells (C) were used as positive controls.

Supplementary Table1.Candidate miRNAs whose expression levels were altered more than 1.5-fold in Panc1-GRs compared with Panc1-P cells.

Supplementary Table2.Candidate miRNAs whose expression levels were altered more than 1.5-fold in Panc1-P (Sp) compared to Panc1-P cells.

Supplementary Table3. The clinicopathological characteristics of the 24 patients.

Between March 2007 and December 2010, 60 patients underwent surgery for pancreatic cancer at Osaka University Hospital, Osaka, Japan. Among those patients, 24 consecutive patients who underwent curative resection (R0) histologically with no preoperative therapy were enrolled in this study. The characteristics of the 24 patients were summarized.