Additional file 1: Figure S1. of Efficient elimination of pancreatic cancer stem cells by hedgehog/GLI inhibitor GANT61 in combination with mTOR inhibition

Staining of Capan-1 M9 cells without first antibodies as negative controls (A) and with antibody against β-catenin or Notch 1 (intra cellular domain: NICD) (B). Scale bar: 100 μm. Similar results were obtained in the staining of PANC-1, MIA PaCa-2 and Capan-1 cells (data not shown). The experiment was repeated three times and the representative results were indicated. Figure S2. The effects of signaling inhibitors on the frequency of CD133+ cells and the viability of pancreatic cancer cells. We treated Capan-1 M9 cells with the inhibitors at the indicated concentration for 3 days, then determined the frequency of CD133+ cells and their cell viability by flow cytometry and the MTT assay, respectively. The results are presented as percentages of control values in untreated cells, showing the mean and standard deviation (s.d.) of 4 replicates obtained in 1 representative experiment. Figure S3. The effects of GANT61 on the surface markers and the shape of Capan-1 M9 pancreatic cancer cells. (A) Flow cytometric analysis of CD133 and CD44 expression on Capan-1 M9 cells treated with or without GANT61. We treated the Capan-1 M9 cells for 3 days, then analyzed them. (B) Representative photographs of Capan-1 M9 cells untreated and treated with GANT61. GANT61 treatment induced a morphologic change of the Capan-1 M9 cells. This may correlate with the increased baseline level in the flow cytometric analysis of GANT61 treated cells shown in (A), which reflect the changes in the structure of the cell surface. Scale bar: 100 μm. Figure S4. The SMO-agonist SAG does not stimulate the sphere formation in Capam1M9 cells. We treated Capan-1 M9 cells with indicate4d concentration of SAG during sphere formation and determined the number of spheres formed per 96-well plate in triplicate. All data are mean and s.d. in 1 representative experiment out of 2. Table S1.Tumorigenesis of pancreatic　cancer cell lines in NOD/SCID mice. Figure S5. The limiting dilution method is necessary to analyze the sphere-forming ability of PANC-1 cells. (A) PANC-1 cells form large aggregates in the multicellular sphere formation assay. We plated single-cell suspensions (103 cells in 0.5 ml) in 24 wells and cultured them. Representative photographs from day 2. (B) Representative photographs of PANC-1 spheres in the single-cell sphere formation assay. We plated single-cell suspensions (1.2 cells in 100 μl) in 96 wells and cultured them for 8 days. Scale bar: 100 μm. Figure S6. A heatmap of the DNA microarray demonstrates upregulation of GLI1 expression in spheres of Capan-1 M9 cells under stem cell culture condition. M9: Capan-1 M9 cells in two-dimensional culture with 10 % FCS, M9 Sphere: Capan-1 M9 spheres formed in a stem cell culture medium. Figure S7. (A) The hedgehog (Hh)/GLI inhibitor GANT61 (10 μM) in combination with the mTOR inhibitor rapamycin (10 nM) effectively reduced the sphere formation of Capan-1 M9 cells. The results are presented as in Fig. 3c. (B) The hedgehog (Hh)/GLI inhibitor GANT61 in combination with the mTOR inhibitor rapamycin impairs in vivo growth of xenografted pancreatic carcinoma in nude mice. Tumor growth of Capan-1 M9 xenografts which were treated with vehicle, 40 mg/kg of body weight, 1 mg/kg of body weight, or combination. Tumor volume was shown as the mean and SD from 5 mice (10 tumors) from each group. * P < 0.05. Figure S8. A schematic representation of the possible mechanism(s) underlying the synergistic effect of double blockage of Hh and mTOR signaling. The decoupling of GLI activation from SHH-PTCH-SMO signaling and/or the redundancy of canonical and noncanonical GLI activation may underlie the ineffectiveness of Hh/SMO inhibitors. This makes GLI as the key molecule in Hh pathway. The difference in the effect of Hh/GLI inhibitors (which reduces CD133+ cell content) and the mTOR inhibitor (which does not reduce CD133+ cell content) suggests that the mTOR function is not mediated only by S6K. Assuming the specific function of mTOR (*) will explain the different effect. (PDF 466 kb