Figure S1. Isolation of cancer stem cells from pancreatic cancer lines. (A) Aldefluor assay of PK1, PK45H and AsPC1 cells. Cells strongly expressing ALDH1A1 were not isolated by FACS. (B) Aldefluor assay of PANK1, PK4-1 and SUIT-2 cells. Cells strongly expressing ALDH1A1 were isolated using FACS. A specific inhibitor of aldehyde dehydrogenase, DEAB, was used to control for background fluorescence. (C) Sorted SUIT-2 cells strongly expressing ALDH1A1 did not form fine spheres in the sphere formation assay. ALDH1A1, aldehyde dehydrogenase 1 family member A1; DEAB, diethylaminobenzaldehyde. Figure S2. Proliferation of cells exposed to 4-AP, 5-FU or 5-FU combined with 4-AP. Cell counts at 72 h were reduced in 4-AP (2 mM)- or 5-FU (4 µM)-treated PK59 cells. 4-AP enhanced the inhibitory effects of 5-FU. Data are presented as the mean ± SEM. n=3. *P<0.05. 4-AP, 4-aminopyridine; 5-FU, 5-fluorouracil. Table SI. Top 50 displaying the greatest fold-change in expression levels in cancer stem cells isolated from PK59 cells.

A, Upregulated genes

Symbol UniGene ID Name Fold-change

SOX2 Hs.518438 Sex-determining region Y-box 2 12,838.901 BEX3 Hs.448588 Brain-expressed X-linked 3 10,831.251 LDHB Hs.446149 Lactate dehydrogenase B 9,775.18 GNG11 Hs.83381 G subunit γ11 7,003.812 RNF130 Hs.484363 Ring finger protein 130 4,556.775 HGF Hs.396530 Hepatocyte growth factor 4,340.44 MAGEA6 Hs.441113 MAGE family member A6 4,135.644 RNF182 Hs.111164 Ring finger protein 182 3,579.186 PSAT1 Hs.494261 Phosphoserine aminotransferase 1 3,458.866 TCEAL4 Hs.194329 Transcription elongation factor A like 4 3,247.818 ATCAY Hs.418055 ATCAY, caytaxin 3,080.797 CCL2 Hs.303649 C-C motif chemokine ligand 2 2,487.519 C11orf96 Hs.530443 11 open reading frame 96 2,360.453 TFPI2 Hs.438231 Tissue factor pathway inhibitor 2 2,321.628 IGFBP7 Hs.479808 Insulin-like growth factor-binding 2,123.917 protein 7 RFLNB Hs.741149 Refilin B 2,082.689 GNB4 Hs.173030 G protein subunit β4 1,929.638 OLFML2A Hs.357004 Olfactomedin like 2A 1,860.643 GAP43 Hs.134974 Growth-associated protein 43 1,854.405 NETO2 Hs.645802 Neuropilin and tolloid-like 2 1,645.218 CTSF Hs.11590 Cathepsin F 1,489.241 BNIP3 Hs.144873 BCL2-interacting protein 3 1,486.117 GSTM3 Hs.2006 Glutathione S-transferase µ3 1,462.533 TCEAL9 Hs.533287 Transcription elongation 1,353.273 factor A-like 9 VEGFC Hs.435215 Vascular endothelial growth factor C 1,297.124 GLI1 Hs.632702 GLI family zinc finger 1 1,174.283 HRASLS Hs.36761 HRAS-like suppressor 1,172.793 HS6ST2 Hs.385956 Heparan sulfate 6-O-sulfotransferase 2 1,159.224 BASP1 Hs.201641 Brain abundant membrane-attached 1,136.464 signal protein 1 RTN1 Hs.368626 Reticulon 1 1,038.481 DNAH14 Hs.133977 Gynein axonemal heavy chain 14 969.738 SCD5 Hs.379191 Stearoyl-CoA desaturase 5 951.581 CBS/CBSL Hs.533013 Cystathionine-β-synthase 950.998 SCG2 Hs.516726 Secretogranin II 946.758 JAM2 Hs.517227 Junctional adhesion molecule 2 888.524 ADRA1B Hs.368632 Adrenoceptor α1B 872.55 ADGRL3 Hs.570770 Adhesion G protein-coupled receptor L3 865.439 PRAME Hs.30743 Preferentially expressed antigen 848.157 in melanoma CHGB Hs.516874 Chromogranin B 834.502 FAM92A Hs.125038 Family with sequence similarity 92 826.411 member A KIRREL2 Hs.145729 Kirre-like nephrin family adhesion 813.037 molecule 2 MAP9 Hs.61271 -associated protein 9 807.206 HDGFL3 Hs.513954 HDGF-like 3 793.612 ZCCHC11 Hs.655407 Zinc finger CCHC-type containing 11 767.257 RGS20 Hs.368733 Regulator of G protein signaling 20 712.106 LHX2 Hs.696425 LIM homeobox 2 683.548 PHGDH Hs.487296 Phosphoglycerate dehydrogenase 680.689 SALL2 Hs.416358 Spalt-like transcription factor 2 671.161 EXO5 Hs.59584 Exonuclease 5 641.587 GJC1 Hs.712052 Gap junction protein γ1 629.762 Table SI. Continued.

B, Downregulated genes

Exp Symbol UniGene ID Entrez gene name fold-change

TACSTD2 Hs.23582 Tumor-associated calcium signal -24,246.89 transducer 2 CEACAM6 Hs.466814 Carcinoembryonic antigen-related cell -22,946.62 adhesion molecule 6 UCA1 Hs.644234 Urothelial cancer-associated 1 -19,804.604 (non-protein coding) THBD Hs.2030 Thrombomodulin -12,830.747 MAL2 Hs.201083 Mal, T-cell differentiation protein 2 -12,013.591 (gene/pseudogene) KRT6A Hs.700779 6A -10,672.606 CST6 Hs.139389 Cystatin E/M -7,174.807 KRT6C Hs.709234 Keratin 6C -7,044.094 WFDC2 Hs.2719 WAP four-disulfide core domain 2 -7,040.454 ADIRF Hs.642660 Adipogenesis regulatory factor -7,001.022 S100P Hs.2962 S100 calcium-binding protein P -6,682.106 SCEL Hs.534699 Sciellin -5,723.248 FXYD3 Hs.301350 FXYD domain containing ion transport -5,700.655 regulator 3 KRT86 Hs.278658 Keratin 86 -5,564.442 SERPINB5 Hs.55279 Serpin family B member 5 -5,475.957 ANXA3 Hs.480042 Annexin A3 -5,068.805 ANKS6 Hs.406890 Ankyrin repeat and sterile α motif -5,018.736 domain-containing 6 MPZL2 Hs.116651 Myelin protein zero like 2 -4,660.765 H19 Hs.533566 H19, imprinted maternally expressed -3,505.563 transcript (non-protein coding) KLK6 Hs.79361 Kallikrein-related peptidase 6 -3,320.966 LAD1 Hs.519035 Ladinin 1 -3,285.837 FBP1 Hs.494496 Fructose-bisphosphatase 1 -2,831.942 KRT83 Hs.720768 Keratin 83 -2,692.064 PLAT Hs.491582 Plasminogen activator, tissue type -2,639.323 SERPINB1 Hs.381167 Serpin family B member 1 -2,570.951 CEACAM7 Hs.74466 Carcinoembryonic antigen-related cell -2,525.988 adhesion molecule 7 FA2H Hs.461329 Fatty acid 2-hydroxylase -2,474.334 LY6D Hs.415762 Lymphocyte antigen 6 family member D -2,434.687 EFEMP1 Hs.76224 EGF-containing fibulin-like extracellular -2,320.905 matrix protein 1 CACNG6 Hs.631560 Calcium voltage-gated channel auxiliary -2,192.582 subunit γ6 KRT5 Hs.433845 Keratin 5 -2,148.582 ANXA8L1 Hs.744068 Annexin A8-like 1 -2,019.645 B3GNT3 Hs.69009 UDP-GlcNAc:βGal β-1,3-N-acetyl -1,890.112 glucosaminyltransferase 3 SLPI Hs.517070 Secretory leukocyte peptidase inhibitor -1,863.642 TRIM29 Hs.504115 Tripartite motif-containing 29 -1,804.841 TIMP3 Hs.644633 TIMP metallopeptidase inhibitor 3 -1,754.615 RRBP1 Hs.472213 Ribosome-binding protein 1 -1,744.391 CLIC5 Hs.485489 Chloride intracellular channel 5 -1,718.318 TNS4 Hs.438292 Tensin 4 -1,663.067 INPP5D Hs.601911 Inositol polyphosphate-5-phosphatase D -1,637.828 OVOL1 Hs.134434 Ovo-like transcriptional repressor 1 -1,630.057 KLK8 Hs.104570 Kallikrein-related peptidase 8 -1,580.523 Table SI. Continued.

B, Downregulated genes

Symbol UniGene ID Entrez Gene Name Fold-change

MALL Hs.185055 Mal, T-cell differentiation protein-like -1,527.705 IL1RN Hs.81134 Interleukin 1 receptor antagonist -1,483.609 BTBD6 Hs.7367 BTB domain-containing 6 -1,471.619 TMEM139 Hs.731832 Transmembrane protein 139 -1,443.319 MAPK13 Hs.178695 Mitogen-activated protein kinase 13 -1,430.093 PYCARD Hs.499094 PYD and CARD domain-containing -1,402.158 CDKN2A Hs.512599 Cyclin-dependent kinase inhibitor 2A -1,402.143 MGST2 Hs.81874 Microsomal glutathione S-transferase 2 -1,286.587 Table SII. CSC marker-related gene expression levels in CSCs isolated from PK59 cells.

Symbol UniGene ID Entrez Gene Name Fold-change

SOX2 Hs.518438 Sex-determining region Y-box 2 12,838.901 ALDH1A1 Hs.76392 Homo sapiens aldehyde dehydrogenase 1 19.726 family member A1 CXCR4 Hs.593413 C-X-C motif chemokine receptor 4 14.078

CSC, cancer stem cell.