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Fold change in mRNA expression
Figure S1 – Autofluoresence as a biomarker for pancreatic cancer stem cells A Fluo– Fluo+ B Fluo– Fluo+ * NANOG * * β-ACTIN * Fold change in mRNA level Fold change in protein level Fluo– Fluo+ C Panc parental tumor 2.83% 488 (580/30) Single Fluo+ CSC 488 (530/40) Single Fluo+ CSC-derived tumor D * * * Fold change in mRNA expression * * 5.72% * * * * 488 (580/30) 488 (530/40)
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Fold change as compared to control
Figure S2 – PDAC CSCs over-express DNMT1 and bear higher 5mC levels A 185 Fluo– Fluo+ CD133– CD133+ 5mC 1.00 1.30 1.00 1.82 B DNMT1 * Fold change as compared to control Fluo– Fluo+
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Figure S3 – DNMT1 inhibitor Zebularine decreases CSC phenotype
FSC-A Adherent Spheres CD133 8.35% 8.49% 25.9% 29.3% B NANOG A6L * CD133 – + NANOG β-ACTIN Fold change in mRNA expression 1 1.23 DNMT1 185 β-ACTIN 1 1.38 CD133– CD133+ Vehicle * C D DNMT1 * Cytotoxicity (% of control) Fold change in mRNA expression Ctrl Zeb Ctrl Zeb Ctrl Zeb A6L 185 354 Zebularine E Fold change in Fluo+ cells Ctrl Zeb * Ctrl Zeb 6.79% 4.51% 488 (530/40) Fluo+ 488 (580/30)
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Fold change in mRNA expression
Figure S4 – DNMT1 inhibitor Decitabine decreased CSC phenotypes A D Fold change in mRNA expression * Ctrl Dac * Cytotoxicity compared to control Ctrl 0.1mM 0.5mM 1mM 5mM E Decitabine E-CADHERIN FSC-A 15.2% 45.0% 6.39% 32.3% Ctrl B Ctrl Dac DNMT1 GAPDH 1.00 0.32 Dac * C Pan-CK Fold change in protein expression * % of spheres compared to control Ctrl Dac Ctrl Dac 1st generation 2nd generation E-CADHERIN Pan-CK
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Fold change in protein expression
Figure S5 – DNMT1 inhibitor Zebularine promotes CSC differentiation A Pan-CK Ctrl Zeb B Fold change in protein expression Pan-CK Ctrl Zeb 15.2% 33.9% * * FSC-A E-CADHERIN Ctrl Zeb E-cadherin 6.39% 11.3% E-CADHERIN Pan-CK C Cas9 Cas9+sgDNMT1 5.66% 10.9% FSC-A Pan-CK
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Figure S6 – DNMT1 inhibition modulates expression of miR-203, miR-205, and the miR-17-92 cluster
* Fold change in miR expression Fold change in miR expression Ctrl Zeb Cas9 Cas9+sgDNMT1 C * Fold change in miR expression Cas9 Cas9+sgDNMT1
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Table S1 – Decreased DNA methylation of genes upon Zebularine treatment
Gene_Name FC_Zeb vs Ctrl PRR3/GNL1 0.09 LIFR 0.23 UBE2G1 0.27 KIAA1432 0.30 ITSN1/CRYZL1 HIST1H4K 0.33 PITX1 0.34 HIST1H3I HES2 GARNL3 0.35 LOC389333 0.37 UXS1 LANCL1/CPS1 0.38 PCBD2 SSTR5/LOC146336 0.39 PSTK NDUFS6 0.40 ADC ZBTB7A HIST1H2AJ/HIST1H2BM FOXP1 EEF1A1 HDHD2 USP1 0.41 MAPKBP1 ZFP36 0.42 SGK3 TUBB2B GCLC 0.43 SOX2OT/SOX2 SHOX2 UBAC2 ILF3/LOC147727 STX5 0.44 HLA-DRB1 SPEN/FLJ37453 SGEF ECHDC2 HIST1H4J 0.45 MAP6D1 LOC /RAD9A VPRBP 0.46 TMEM179B/TAF6L DOCK1 PTGER3 EFHC1 ANKRD57 TMEM108 0.47 ERICH1 PLEK2 Gene_Name FC_Zeb vs Ctrl BCAR1 0.48 ICT1 RAB6A TCTE1 MYADM ELAC2 EIF3I ZNF784 DPP3 TRIM24 BCCIP PSTPIP2 PRKAG2 LYST DUSP1 C1orf210 CBR1 SIN3A KIAA0802 0.49 NDE1 C21orf96 ALKBH5 FRAS1 ANKH IFNGR1 ATP1A1 KCNT1 CDC6 LEPR/LEPROT SLC7A6 EZH2 DCP1A HNRNPD C2orf63/RPS27A PCDHGA1 GPX5 BAT2L2 0.50 HIST2H2AA3 FAM119A MTCH1 FDX1L TNPO3 C7orf55 AS3MT UNC84A
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Table S2 – Increased DNA methylation of genes upon Zebularine treatment
Gene_Name FC_Zeb vs Ctrl LRP11 9.51 HOXA3 9.20 CMTM3 7.71 TRMT11 7.27 SFRS2IP 5.99 LYN 5.06 EXOC4 3.72 TESK2 3.65 RNF168 3.59 SIVA1 3.54 FKBP2 3.42 TMEM160 3.17 LPHN2 2.85 METT10D CHD9 2.84 RAD51 ESYT1 2.76 NFYA/C6orf130 2.74 H2AFY2 2.73 NAV1 2.72 BLZF1/NME7 2.71 C6orf192 2.70 KCTD6 SYCP3 2.69 UTRN NEDD9 2.68 FTO/RPGRIP1L BAI2 ZNF518A 2.67 BRCA2 2.66 MAPKAP1 2.65 SNORD50A/SNHG5 2.64 ATN1 2.63 TPM3 KCTD11/ACAP1 MAP7 2.62 SF3B1 SLC35A1 2.61 ANO4 DAXX KIF11 2.60 ZNF613 NCOA6 2.58 ABCF1 MACC1 SNHG3-RCC1 2.57 ZDHHC20 2.56 C18orf54 WHSC1L1 2.55 HIST2H2AA4/HIST2H2AA3 RFC3 DAPK3 RB1CC1 2.53 LARS2 2.52 Gene_Name FC_Zeb vs Ctrl DCUN1D4 2.51 PCDHB10 APP 2.50 ALDH1A3 MIR548I4/CNTNAP2 2.49 MEF2D TRIM25 2.48 IFNAR2 2.47 KLC4/MRPL2 CLUL1 DTL 2.46 MAP3K5 OXCT1 2.45 HIST1H4E FDFT1 TMED10 UGDH PJA2 2.43 EIF3H PSMC2 SOCS4/WDHD1 OR5I1 ELL2 2.42 GRB2 FUK ATP5J/GABPA 2.41 HIST1H2BM/HIST1H2AJ BAT4/CSNK2B SOX2OT/SOX2 2.40 CTNND1 SYDE2 SFRS12IP1 NMI NCRNA00120/AKIRIN2 2.39 PROX1 USP13 BAI3 RBM47 GLI1 2.38 RAP2A RPLP2 NAA16 CDC14B 2.37 MYO16 PTP4A2 BET1 C1orf25 SYNE2 FLJ45244/DICER1 2.36 HOXC11 HSPC072/LOC 2.35 PSMA1 GTF3A
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Table S2 – Increased DNA methylation of genes upon Zebularine treatment
Gene_Name FC_Zeb vs Ctrl RNASEK 2.35 C10orf53 CD2AP PSMG2/CEP76 2.34 KIAA1244 DCP2 CNN3 BZW2/ANKMY2 2.33 DLAT SLC7A11 BTN2A1 RAD54B C6orf165 CYBASC3/TMEM138 GNAO1 2.32 MRPS30 SIN3A SLC2A1 ZNF259 HIST1H2AC/HIST1H2BC 2.31 B3GALT1 RPL14 API5 TMEM87A 2.30 MCM4 NDC80/METTL4 ATF6B PTPRD MAP4K1/EIF3K BRE/RBKS/LOC MTRF1L 2.29 SC4MOL TOLLIP LAMB2 NT5DC3 CBR4 PRLR ARL5A PLK1 2.28 IWS1 SLC12A9 HNRNPC NRP1 NMT1/DCAKD IL17RD 2.27 DAB2 LOC552889 UFD1L/CDC45L CDK6 TSNAX-DISC1 2.26 SEPHS2 TMED7-TICAM2 KRT10/TMEM99 RNF111 Gene_Name FC_Zeb vs Ctrl RPL7/RDH10 2.26 C5orf44/TRIM23 IMPAD1 2.25 HARS SLC25A13 CARD6 KIAA0146 PES1 2.24 ZMYND11 GTPBP8 HARBI1/KIAA0652 C12orf11/FGFR1OP2 UBR5 2.23 S100A10 TMEM49/PTRH2 VWC2 FSIP1 MRPS28 2.22 HAX1 LRRK1 CASC5 BRIX1/RAD1 C7orf36 ALK ZDHHC5 2.21 ADAR LENG9 NR1H3/ACP2 LARP7/C4orf21 MAP4 PHF12 IPMK VPS25 C2orf60 2.20 NFIL3 KIAA0319L TBC1D4 CBARA1 MAGI1 TP53BP1 2.19 MIPOL1 SLC1A3 C17orf80/FAM104A MADD HIST1H1E 2.18 ALG14 SACM1L MRFAP1L1 ALCAM MED7 DTWD2 DLG2 SLC25A36 ZNF773 2.17
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Table S2 – Increased DNA methylation of genes upon Zebularine treatment
Gene_Name FC_Zeb vs Ctrl RAI14 2.17 BRD2 C14orf106 KLF9 C11orf65 NUP98 MRPS12/SARS2 NUF2 CHD3 RRM2B COX7A2 SERPINI1/PDCD10 2.16 MED27 C16orf71/ANKS3 UFD1L/CDC45L BTAF1 LHFP ZRANB2 TRIM26 POLR1A AGPAT1/RNF5/RNF5P1 CCDC94 ZBTB9 DERL2/MIS12 2.15 MIR548G/C3orf26 ERGIC2 LOC ATF4 SKA3/MRP63 ARID2 ANKRD34A/POLR3GL DEFA5 MAT2B ZNF687 CWC22 2.14 MALAT1 C4orf33 LOC /TPT1 LIPH EIF3B MLX TMCO6 SPATA17/GPATCH2 PNPLA3 C12orf76 BAT3 2.13 RPS27 KDM1A PLEKHM1 FBXW8 SDAD1 PGAP2/NUP98 PSMB8 SRPK1 Gene_Name FC_Zeb vs Ctrl C6orf52/PAK1IP1 2.13 ETAA1 HSD11B1L/C19orf70 ATF2/MIR933 DLGAP5 HIVEP3 ALG14 HERC4 2.12 MRPS33 C21orf54 TNFRSF11A C16orf46 RPL22 MRPS14 C10orf131 HFE ADPRHL2 PSPC1 ARGLU1 2.11 RAB12 CAPZA1 LRCH3 TMTC2 LOC285830 THNSL1/ENKUR HIST1H2AB VTI1A/ZDHHC6 USP39 FAM109A TLE4 TSN ROBO3 C10orf114 2.10 SV2C NECAB1 TFIP11 DCBLD2 IPO11 ZNF219/C14orf176 PRIM2 HNF1A C13orf38 C12orf72 FLJ43663 MLH1/EPM2AIP1 MRPS36 HSBP1L1 ANKFY1 NCEH1 PAPOLG 2.09 LMBR1 ZNF423
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Table S2 – Increased DNA methylation of genes upon Zebularine treatment
Gene_Name FC_Zeb vs Ctrl WDR89 2.09 HIST1H1D RPS11 SP2 SNW1/C14orf178 KIAA0319 EHBP1 ZBTB4/POLR2A TMEM68/TGS1 IMMT HM13 TMF1 INSIG2 OSTC LPHN1 PPP2R3C/KIAA0391 PODNL1/DCAF15 C7orf64/PEX1 LSR ZNF184 VEZF1 ZNF705A/FAM66C ACCN1 DLX2 CHD7 ANO4 ABCF1 UNC45A 2.08 ELP3 ZNF440 CEP164 KAT2B CYB5A CDV3 NME7/BLZF1 FKBPL PRNP ZNF426 SLK KCTD21/USP35 EHMT2 APOC1 SAPS2 2.07 TPI1 ASNSD1 TMEM143/SYNGR4 KLF9 C2orf60 CASC2/RAB11FIP2 AHCYL1 C13orf23/NHLRC3 ZNF192 AGPAT1/RNF5/RNF5P1 CHURC1 Gene_Name FC_Zeb vs Ctrl ATP13A3 2.07 VPS45 KIAA0907 SETD2 ORC1L/PRPF38A BNIP1 SFRS13A OSBP MPZL2 WDR46/PFDN6 KIAA0427 ZFYVE26 HIST1H2AC OGG1 HELQ/MRPS18C MXI1 SYF2 TOMM7 PTEN/KILLIN RNGTT NPC2 CENPO KRAS 2.06 RPUSD2 RGS2 SORL1 LOC388789 C1orf204 C4orf3 RPL24 UBXN2A PRKG1 MRPS36 SETD5 MEPCE/ZCWPW1 CA8 RPL7 ITGAV MET FCF1/KIAA0317 DDX18 DPYD VTI1B CD8A SLC39A7/RXRB 2.05 RPS6KC1 SMARCAD1 HOXA3 CTDSPL2 KCTD18 HIST1H2BN CCND3 SCARNA16/C17orf86 RSL1D1
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Table S2 – Increased DNA methylation of genes upon Zebularine treatment
Gene_Name FC_Zeb vs Ctrl BCO2 2.05 C1orf31 RUNX1 CD55 KIAA1522 TIMELESS CCDC18/TMED5 ATOX1 2.04 TACO1 NAPB PEX7 C4orf32 NCRNA00081/SHOC2 WBP4 TUFM TRIM27 IRF2BP2 RTTN HNRNPA1/CBX5 EIF3E MOBKL1A ERCC5 CRAMP1L ATP5G1 RBPJL/MATN4 PFDN6/WDR46 GLCCI1 S100A11 2.03 FAM102B SFPQ HIST1H2AB SNORA7A/RPL32 HNRNPM SLC35E3 ANKRD11 BUB1 OR1E2 ASAH1 DARS CTDSP2 PTTG1IP TMX4 ANAPC7 YARS RND3 GBX1 OSGEPL1 2.02 CACNA2D3 BAD/GPR137 NRD1 TRIM2 TRPM5 MACF1 NUAK2 Gene_Name FC_Zeb vs Ctrl KBTBD5 2.02 C10orf140 BDP1 LANCL1/CPS1 DPYSL2 HIP1 ZADH2/TSHZ1 ROBO1 FBXW8 UBR4 IFI16 BTBD9 ACAD11 DTNBP1 PCDH7 ZFP36L2/LOC PEX13 MAF HIST1H2AG/HIST1H2BJ RASL11B ORC6L/VPS35 RAB13 2.01 SLK SPRY1 CCDC49 CCDC21 CCDC14 FBXL3 KLHDC4 TRIM45 TBP AICDA RFXANK NUP188/ FBXO16 UBP1 GNAI3 A2BP1 AASDH GPM6A UBN2 PIK3R3 TAF11/ANKS1A LSG1 TMEM67 MSH5 NPM1 C9orf130 2.00 RPL32P3 SCGB2A2 YWHAG CDC42EP3 C19orf35 EXOSC9 Gene_Name FC_Zeb vs Ctrl NQO1 2.00 ALS2CR4 ANKRD10 MTFMT MED17 ARMC8 MTX2 ANGEL2
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Table S3 – Decreased DNA methylation for miRs following Zebularine treatment
hsa-miRNA FC_Zeb vs. Ctrl hsa-miR1470 0.61 hsa-miR548G 0.69 hsa-miR205 0.71 0.73 0.74 hsa-miR548N 0.77 0.78 0.80 hsa-miR1236 0.58 hsa-miR548H4 0.62 hsa-miR636 hsa-miR203 hsa-miR26B 0.70 0.72 hsa-miR423 0.75 hsa-miR219-1 0.76 hsa-miR148A 0.79 hsa-miR760 hsa-miR1915 hsa-miR1324 0.63 hsa-miR2277 0.67 hsa-miR15B/miR16-2 0.68 hsa-miR548F1 hsa-miR2110 hsa-miR346 hsa-miR92B hsa-miR654/miR376B/miR376A2/miR300/miR376A1 hsa-miR1292 hsa-miR1322 hsa-miR1539 hsa-miR611 hsa-miR129-1 hsa-miR564 hsa-miR132/miR212 hsa-miR1205 hsa-miR301B/miR130B hsa-miR548H3 hsa-miR600 hsa-miR27A/miR24-2/miR23A
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Table S3 – Decreased DNA methylation for miRs following Zebularine treatment
hsa-miRNA FC_ Zeb vs. Ctrl hsa-miR202 0.75 hsa-miR570 hsa-miR567 hsa-miR585 0.76 hsa-miR592 hsa-miR375 hsa-miR339 0.77 hsa-miR191/miR425 hsa-miR150 hsa-miR130A hsa-miR17HG 0.78 hsa-miR658 hsa-miR149 hsa-miR661 hsa-miR548K hsa-miR1304 hsa-miR574 hsa-miR320A 0.79 hsa-miR16-2 hsa-miR30D hsa-miR1977 hsa-miR190B hsa-miR638 hsa-miR425 hsa-miR758/miR1197/miR329-1/miR323 hsa-miR596 hsa-miR451/miR144 hsa-miR10A 0.80 hsa-miR639 hsa-miR1252 hsa-miR10B hsa-miR762 hsa-miR1287 hsa-miR141/miR200C hsa-miR26A2 Given is the Fold Change (FC) of β-methylation values for Zebularine (Zeb) treatment vs control (ctrl). Only data for FC ≤ 0.80 are provided. Due to the presence of different probes targeting the same miRNAs, some are shown more than once.
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