SUPPLEMENTARY INFORMATION. Supplemenary Figures Figure S1
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1 doi: /nature05939 SUPPLEMENTARY INFORMATION Supplemenary Figures Figure S1 wt MEF.1 wt MEF.2 wt MEF.Myc.1 wt MEF.Myc.2 wt MEF.Ras.1 wt MEF.Ras.2 wt MEF.E1A/Ras.1 wt MEF.E1A/Ras.2 p53-/- MEF.E1A/Ras.1 p53-/- MEF.E1A/Ras.2 p53-/- MEF.1 p53-/- MEF.2 p53-/- MEF.Ras.1 p53-/- MEF.Ras.2 p53-/- MEF.Myc.1 p53-/- MEF.Myc.2 Figure S1. mirna profiles of engineered MEFs. The full heatmap for the unsupervised clustering of MEF lines, with the indicated genotypes, corresponding to Fig 1A, is shown. Values are shown on a linear scale. let-7d mir-339 let-7g mir-26a let-7c mir-378 mir-210 mir-130a mir-301 mir-15a mir-16 mir-15b mir-345 let-7i mir-130b mir-19b mir-19a mir-7 mir-17-5p mir-20 mir-106a mir-18 mir-92 mir-298 mir-30c mir-32 mir-106b mir-93 mir-26b mir-30a-5p mir-30b mir-196b mir-196a mir-101 mir-195 mir-361 mir-126* mir-340 mir-186 mir-182 mir-183 mir-96 mir-148b mir-331 mir-194 let-7e mir-191 mir-328 mir-200c mir-34b mir-34c mir-34a mir-27a mir-98 mir-103 mir-135b mir-31 mir-29c mir-29a mir-222 mir-29b mir-30d mir-30e mir-341 mir-425 mir-206 mir-212 mir-188 mir-142-5p mir-150 mir-320 mir-342 mir-132 mir-149 mir-218 mir-25 mir-148a mir-152 mir-181a mir-181c mir-133b mir-181b let-7b mir-27b mir-23b mir-335 mir-10a mir-189 mir-154 mir-296 mir-193 mir-107 mir-125a mir-125b mir-21 mir-22 mir-99b mir-28 mir-135a mir-23a mir-24 mir-199a mir-214 mir-100 mir-145 mir-143 mir-380-5p mir-300 mir-337 mir-329 mir-382 mir-323 mir-299 mir-146 mir-223 Fold Change
2 . doi: /nature05939 SUPPLEMENTARY INFORMATION Figure S2 hsa-mir-34a - UGGCAGUGUC - UUAGCUGGUUGUUGUGAGCAAUAGU - A mmu-mir-34a - UGGCAGUGUC - UUAGCUGGUUGUUGUGAGUAUUAGCUA gga-mir-34a - UGGCAGUGUC - UUAGCUGGUUGUUGUGAGCAAUAGUUA dre-mir-34 - UGGCAGUGUC - UUAGCUGGUUGUUGUGUGGAGUGAG - A hsa-mir-34b UAGGCAGUGUCAUUAGCUGAUUGUACUGUG - - GUGGUUA mmu-mir-34b UAGGCAGUGUAAUUAGCUGAUUGUAGUGCG - - GUGCUGA gga-mir-34b - AGGCAGUGUAGUUAGCUGAUUGUACCCAGCGCCCCA - - dre-mir-34b UAGGCAGUGUUGUUAGCUGAUUGUUUCAUAUGAACUA - - hsa-mir-34c - AGGCAGUGUAGUUAGCUGAUUGCU - - AAUAGUAC mmu-mir-34c - AGGCAGUGUAGUUAGCUGAUUGCU - - AAUAGUAC gga-mir-34c - AGGCAGUGUAGUUAGCUGAUUGCC - - ACCAGGAC dre-mir-34c - AGGCAGUGCAGUUAGUUGAUUACA - - AUCCAUAAAG - - dme-mir-34 - UGGCAGUGUGGUUAGCUGGUUGUGUAGCCAAU - UAUUG dps-mir-34 - UGGCAGUGUGGUUAGCUGGUUGUGUAGCCAAAAUAUUG cbr-mir-34 - AGGCAGUGUGGUUAGCUGGUUGCAUACACAGG cel-mir-34 - AGGCAGUGUGGUUAGCUGGUUGCAUAUUUCC hsa-mir-34a A GGAAGCAAUCAGCAAGUAUACUGCCCUA - mmu-mir-34a A GGAAGCAAUCAGCAAGUAUACUGCCCUA - gga-mir-34a A GGAAGCAAUCAGCAAGUAUACUGCCCUA - dre-mir-34 A CGAAGCAAUCAGCAAGUAUACUGCCGCA - hsa-mir-34b CAAUCACUAACUCCACUGCCAUCA mmu-mir-34b CAAUCACUAACUCCACUGCCAUC - gga-mir-34b CAAUCACUAAAUUCACUGCCAU - - dre-mir-34b UAAUCACUAACCAUACUGCCAACA hsa-mir-34c CAAUCACUAACCACACGGCCAGG - mmu-mir-34c CAAUCACUAACCACACAGCCAGG - gga-mir-34c CAAUCACUAACCACACAGCCAGG - dre-mir-34c UAAUCACUAACCUCACUACCAGG - dme-mir-34 CCGUUGACAAUUCACAGCCACUAUCUUCACUGCCGCC - dps-mir-34 CCUUUGACCAUUCACAGCCACUAUCUUCACUGCCGCC - cbr-mir UUGACAACGGCUACCUUCACUGCCACC - cel-mir UUGACAACGGCUACCUUCACUGCCACC - Figure S2. Conservation of mir-34. mir-34 represents a family of evolutionarily conserved mirnas, with single conserved homologues in invertebrates, such as flies and worms. 2
3 Figure S3 A fold induction pri-mir-34a pri-mir-34b/34c mp21 day 0 day 1 day 2 day 4 day 6 day 8 B mir34a (copies/cell) WT p53shrna WT p53shrna HEPG2 TOV21G Figure S3. Correlation of mir-34s expression with p53 status. A. Quantification of primary mir-34a and mir-34b/c transcript in MEFs following p53 reactivation. In these cells, p53 is suppressed by a conditional p53 shrna, whose expression was repressed at day 0 by addition of doxycycline. All Ct values are normalized to Ct for day 0. B. TOV21G and HEPG2 cells were engineered to contain a constitutively expressed p53 shrna or a control vector. Shown is the level of mir34a in each of the lines (see Methods). Measurements of mir34b and c in these lines indicated that these family members are not expressed in either the control or shrna line. In A and B, error bars represent the standard error from the mean in separate measurements. 3
4 Figure S4 A B 15 Fold induction pri-mir-34a pri-mir-34b/34c Fold induction 10 5 pri-34a pri-34b/c p21 0 UNT IR 4h IR 8h UNT IR 4h IR 8h wt mice p53-/- mice 0 0h 4h 6h 8h 12h 24h 0h 4h 6h 8h 12h 24h wt MEFs + Adr p53-/- MEFs + Adr C 15 Fold induction 10 5 mature 34a mature 34b mature 34c 0 UNT 4hr 6hr wild type MEFs + Dox 8hr 12hr 24hr UNT 4hr 6hr 8hr 12hr p53 -/- MEFs + Dox 24hr 4
5 Figure S5 15 expression level 10 5 mir-34a mir-34c 0 IMR90+V IMR90+34a IMR90+V IMR90+34b/c Figure S5. Expression levels of mir-34s. Levels of mature mir-34a or mir-34c were measured by Q-PCR in IMR90 cells infected with a control vector or with vectors directing the expression of primary transcripts for mir-34a or mir-34b/c, as indicated. 5
6 Figure S6 A A % 4.9% 30.7% 42.3% 43.4% 2N 4N 2N 4N 2N 4N 2N 4N 2N 4N mock Luc si mir-34a mir-34b mir-34c B 10.7% 11.8% 51.2% 32.2% 39.6% Hct116 p21 -/- Hct % 12.2% 44.0% 43.5% 30.8% 2N 4N 2N 4N 2N 4N 2N 4N 2N 4N mock Luc si mir-34a mir-34b mir-34c Figure S6. mir-34 induces cell cycle arrest in cancer cells. A. A549 cells were transfected with mir-34s as sirnas and cell cycle arrest was measured using a G2 trapping assay. Briefly, cells were treated with Nocodazole 24 hours post transfection and monitored for cell cycle distribution hours after Nocodazole treatment. Proliferating cells accumulate in G2 phase, and only cells arrested in G1 or S prior to Nocodazole treatment retain those states. B. Hct116 and Hct116 p21-/- cells were transfected with mir-34s as sirnas, and cell cycle arrest was monitored as decribed in A. 6
7 Figure S7 Adriamycin down-regulated Adriamycin up-regulated GO category "cell cycle" p-value e mir-34 up-regulated p-value 1.8e e -16 mir-34 down-regulated Figure S7. Functional annotation of candidate mir-34 targets. Venn diagrams summarize functional analysis of mir-34a, b and c consensus signatures across multiple cell types (see Table S2). This analysis reveals the following significant gene set overlaps: mir-34 down-regulated genes with genes downregulated by adriamycin-induced DNA damage in A549 cells (bottom left); mir-34 up-regulated genes with genes up-regulated by adriamycin-induced DNA damage in A549 cells (top middle); mir-34 down-regulated genes with genes in the GO Biological Process category "cell cycle" (bottom right). The p-values for each overlapping set are indicated. 7
8 Figure S8 A A549 HCT116 TOV21G DLD-1 Luc mir34a mir34b mir34c Luc mir34a mir34b mir34c Luc mir34a mir34b mir34c Luc mir34a mir34b mir34c Cell cycle genes enriched (p < 3.4e-20) Log (Ratio) B CCNE2...CCUAGCCAAUUCACAAGUUACACUGCCA... MET...UGUCCAAUGGUUUUUUCACUGCCU... UUGUUGGUCGA------UUC----UGUGACGGU UUGUUGGUCGAUUCUGUGACGGU CDK4...AGUGAGCAAUGGAGUGGCUGCCA... UUGUUGGUCGAUUCUGUGACGGU...CUAAGAAUUAGAUACUUGUCACUGCCU... UUGUUGGUC----GAUUCUGUGACGGU Figure S8. Genes regulated by mir-34. A. RNA duplexes corresponding to each mir34 family member or a control sirna target luciferase (Luc) were transfected into A549, HCT116 Dicerex5, TOV21G, DLD-1 Dicerex5 cells. Total RNA was isolated 24 hrs post transfection, and subjected to microarray expression analysis. Consensus expression signatures that were down-regulated by all mir-34 family mirnas were indicated below the heat map. These consensus signatures were tested for enrichment of cell cycle regulation genes annotated with GO Biological Process terms and 3 UTR hexamer seed matches (see methods). B. Seed hexamer matches from each of the candidates examined in Figure 4 are shown. 8
9 Supplementary Tables and Figure Legends Supplementary Table 1 Table S1. Induction of mirnas and mirnas* by Adriamycin in TOV21G cells copies per cell a microrna induction (fold) untreated Adriamycin mir-34c mir-34b mir mir-302a mir mir mir-34a mir mir mir mir mir-29b mir mir mir mir-182* mir-135a mir mir-130a mir-30a-5p mir-29a mir-30e-3p mir mir mir mir mir mir mir-23a mir mir-30d mir mir mir-133b mir-27a mir-133a mir mir-30e-5p mir-23b mir-10b
10 mir-30c mir-27b mir mir mir-181a mir-26a mir-30b mir mir mir-26b mir-148a mir mir-148b mir-181c mir mir mir-125a mir-125b mir mir-99a mir-106b mir-135b mir mir-15a mir mir mir mir mir mir-10a mir-128b mir mir-19b mir-130b mir-19a mir mir-15b mir mir-9* mir-99b mir mir mir-17-3p mir-128a mir mir mir-106a mir mir mir
11 mir-17-5p mir mir mir mir mir mir mir mir mir-302b mir mir-302d mir mir mir mir-380-3p mir-302c* mir mir-199b mir-199a* mir mir mir-380-5p mir-199a mir mir mir-324-3p mir mir-302a* mir mir mir-200a mir mir mir mir mir-324-5p mir mir-373* mir-196a mir mir mir-196b mir-200b mir mir mir mir mir mir
12 mir mir mir mir mir mir-154* mir mir-422b mir mir mir mir mir mir mir mir mir mir mir N/A mir N/A mir N/A mir N/A mir-422a 0 0 N/A mir N/A mir N/A mir-376b 0 0 N/A mir N/A mir N/A mir N/A mir N/A mir N/A mir N/A mir N/A mir N/A mir-302c 0 0 N/A mir-302b* 0 1 N/A mir N/A mir N/A mir N/A mir N/A mir N/A mir N/A mir N/A mir-142-3p 0 0 N/A mir N/A mir N/A mir N/A mir N/A mir-126* 0 36 N/A mir-124a 0 2 N/A 12
13 mir-122a 0 1 N/A mir N/A a Copies per cell of 194 mirnas and mirnas are determined using real time PCR analysis 1. ΔCt values were converted to copy number by comparison with standard curves generated by use of defined inputs of single stranded mature mirnas. 13
14 Supplementary Table S2 Table S2. Expression alterations for mir-34 downregulated genes in Hct116 Dicer Ex5 cells a Gene Names Accession # b Luc si mir-34a mir-34b mir-34c TK1 NM_ PHF19 AL MET AK LOC BC MCM3 NM_ FLJ11029 AW SH3GL1 NM_ FGFRL1 NM_ CHES1 NM_ PPP1R11 NM_ MGC5508 NM_ CDK4 NM_ C1orf19 NM_ NUP210 NM_ RAB21 BC SLC35A4 NM_ NASP NM_ ANKRD40 AK MGC5242 AK SGPP1 AI LMAN2L NM_ ULBP2 NM_ FKSG24 NM_ CNOT6 NM_ CAP1 NM_ MGC16207 BC FLJ11029 NM_ E2F2 AF TPD52 NM_ TTC19 NM_ GLRX5 NM_ MYB NM_ ATG9A NM_ VAMP2 NM_ SLC29A1 NM_ FAM64A NM_ CDCA5 NM_ CDC25A AI FURIN NM_ DTL NM_ TMED8 AK SHCBP1 NM_ TRIB3 NM_ MET NM_
15 RKHD2 NM_ GMNN NM_ ARHGAP1 NM_ PKMYT1 NM_ MGC13170 NM_ C6orf89 AJ TSPAN14 NM_ FLJ13912 NM_ CDK6 AI MAP3K11 NM_ CTDSPL NM_ CDS2 AI SLC44A2 NM_ TGIF2 NM_ MYOHD1 NM_ CTDSP2 NM_ SURF4 NM_ YKT6 NM_ CDC23 NM_ GNPDA1 NM_ NAGPA NM_ RDH11 NM_ IMPDH1 NM_ SPBC25 NM_ SPFH1 NM_ PHGDH NM_ CHES1 NM_ CCNE2 NM_ XBP1 NM_ RAD54L NM_ RDX NM_ FLJ14154 NM_ SIX5 NM_ FANCA NM_ KIAA1333 NM_ C8orf55 NM_ MGC21644 NM_ TMEM48 NM_ FANCG NM_ CPSF6 NM_ CCNE2 NM_ MCM5 NM_ CTDSP1 NM_ DKFZp564K142 NM_ AXL NM_ KIAA0101 NM_ STMN1 NM_ TAF5 NM_ MBD3 AL FBXO10 BC
16 C7orf21 NM_ HMMR NM_ UBE2L3 NM_ SGPP1 NM_ MYBL2 NM_ RPAP1 NM_ MGC5242 NM_ LASS2 NM_ VPS4A NM_ ZDHHC16 NM_ LRRC40 NM_ C9orf140 NM_ WDR76 AI MGC23280 NM_ UNC84B NM_ VCL NM_ SNX15 NM_ ARAF NM_ C20orf100 NM_ CUEDC1 AI BRCA1 NM_ SFRS1 AI TSN NM_ CUEDC1 NM_ GAS2L3 NM_ ZNF358 NM_ HTLF AA SCRIB NM_ DKFZP564O0823 AK GSG2 NM_ WDR62 NM_ GOLPH3L NM_ PER2 NM_ FEN1 NM_ ERO1L AK CD151 NM_ C6orf89 AK ZNF395 NM_ HMGN4 NM_ EME1 NM_ RP13-15M17.2 AI CIC NM_ MBD3 NM_ KIAA1704 AB AXL NM_ PSF1 D BRRN1 NM_ SLC45A3 NM_ CASKIN2 NM_ CHAF1A NM_
17 RASSF5 NM_ F8 NM_ MGC12538 AA C9orf125 AJ RAD51 NM_ HDAC1 NM_ NFYC NM_ HIST1H4E NM_ PLK1 NM_ PTP4A2 NM_ LOC AL TOM1L2 AL FEM1A NM_ TESK1 NM_ UBE2Q1 NM_ ESPL1 NM_ RRM2 BC SCMH1 NM_ SFXN5 NM_ MTA2 NM_ SURF5 NM_ SLC16A4 AK FUT8 NM_ DTYMK NM_ ATP1B3 NM_ SPBC24 NM_ FLJ37034 BC FLJ13868 NM_ BCL2 NM_ CKLF AI C10orf38 AL CABLES2 BC FLJ39827 NM_ MDM4 NM_ FAM100B NM_ ZDHHC12 NM_ KIAA1160 NM_ ACSL4 NM_ ZHX2 NM_ KIF11 NM_ GTSE1 NM_ DDX10 NM_ NQO1 NM_ ORC1L NM_ PURB AK FLJ14166 NM_ TBC1D13 NM_ PMF1 NM_ IFRD2 NM_ AFG3L1 NM_
18 CEP55 NM_ MKI67 NM_ PLAGL2 NM_ VCL NM_ ARHGDIB NM_ UBE2C NM_ KCNS3 NM_ CCDC15 NM_ LASS5 NM_ PALLD NM_ AREG NM_ PTTG3 NM_ BIRC5 NM_ UBE2C NM_ ABR NM_ ZNF580 NM_ PHF17 NM_ NMT1 NM_ PHB NM_ Pfs2 NM_ NDP52 NM_ DKFZp762E1312 NM_ C9orf10OS AK DDX11 NM_ GCH1 NM_ RNF38 NM_ FSHPRH1 AI LOC AI PARP16 NM_ MAPK9 AI C14orf94 NM_ MPP2 NM_ FAM49B AA HPCAL4 NM_ WHSC1 NM_ C15orf21 NM_ MFN2 NM_ LOC AL ORC6L NM_ QDPR NM_ POLQ NM_ KIF15 NM_ GRPEL2 NM_ FLJ20255 NM_ ZNF395 NM_ HMGB3 NM_ UBP1 NM_ WHSC1 NM_ TATDN2 NM_ HIRIP3 NM_
19 ZNF551 NM_ TUBA2 NM_ ATPAF1 AL RANBP10 AB MAC30 NM_ HIP2 AL CAV1 AF EXOSC2 NM_ ASXL1 NM_ AI KIAA1160 AK TUBAP NG_ MED8 NM_ CDK6 AK KIFC1 NM_ RP13-360B22.2 NM_ EXO1 NM_ EFNA5 AW CCND3 NM_ MAP2K1 NM_ FAM76A AI C9orf25 NM_ W BARD1 NM_ ADRBK2 BC CDC25C NM_ FLJ20232 NM_ BC NDRG1 NM_ PSMB7 AJ D4ST1 NM_ CCNF NM_ CDKN3 NM_ PRR3 NM_ FADS2 NM_ FANCE NM_ CAV1 NM_ SAMD6 NM_ BID AK FIGNL1 NM_ CENPF NM_ DKFZp586I1420 NM_ E2F8 NM_ SLC7A1 AL HCN3 AB KIF20A NM_ DGKZ NM_ DCLRE1B NM_ DHCR24 NM_ ETEA NM_
20 PHF6 NM_ CDC45L NM_ C8orf30A NM_ HMGB3 BC RARG NM_ NUSAP1 NM_ ASF1B NM_ MMS19L NM_ ACSL4 NM_ TRAF7 NM_ C15orf42 NM_ CDCA8 NM_ UHRF2 NM_ FOXM1 NM_ C22orf18 NM_ EVI5L NM_ AADACL1 NM_ ATP1B3P1 NG_ TRIOBP NM_ FUT8 NM_ IQGAP3 NM_ METTL1 NM_ OATL1 L WSB2 NM_ ETV5 NM_ C21orf63 NM_ ENST ENST TBPIP NM_ VDR NM_ FKBP1B NM_ CSRP1 NM_ RRAS NM_ BTRC NM_ IRAK1 NM_ MTMR9 NM_ FBXO5 NM_ MGAT2 NM_ CHMP7 NM_ R3HDM1 NM_ FLJ32363 BC Ells1 NM_ MGC13024 NM_ FOXJ2 NM_ PBEF1 NM_ H2AFX NM_ TESK2 NM_ OXSR1 NM_ RAD51C NM_ RIC8B NM_ KLHDC3 NM_
21 RBM12 NM_ DGAT1 NM_ STX1A NM_ GSK3B AW MKL1 NM_ LASS2 NM_ MLF1IP NM_ SCNN1A NM_ PRC1 NM_ USP3 AK FLJ39660 NM_ PPARG NM_ EIF2AK1 NM_ TMEM22 NM_ HSPC142 NM_ C10orf26 AK C6orf106 NM_ SMPD1 NM_ RRM1 NM_ MSH6 NM_ PPIG R KIF22 NM_ USP15 NM_ LOC AW PTTG1 NM_ PPM1A BM ST3GAL5 NM_ CENPJ NM_ S100A2 NM_ PPRC1 NM_ LOC AL FLOT2 NM_ CDC7 NM_ KIAA0157 NM_ No annotation AK FUT8 NM_ SENP1 BC TNFRSF1A NM_ ARSB AK TTK NM_ KIAA0984 AB RFC4 NM_ CLSPN NM_ AOC3 NM_ PSRC1 NM_ CREB3L2 AL No annotation AI MAP3K7IP2 NM_ C18orf24 NM_ STK39 NM_
22 KIAA0476 NM_ GRK6 NM_ FARP1 AK FLJ22794 NM_ MGC18216 NM_ WHSC1 NM_ TROAP NM_ PRIM1 NM_ TMEM55A NM_ LSS NM_ PURB AK LOC AF BLM NM_ LONRF2 AL No annotation AI KLC2 NM_ STCH NM_ PTTG2 NM_ GDPD5 NM_ CRTC2 NM_ DCTN5 NM_ POU2F1 NM_ KIF4A NM_ ESAM NM_ JPH1 NM_ OVOS2 NM_ ATF4 NM_ CKLF NM_ NT5E AA SLC12A2 AK hcap-d3 D LMNB1 NM_ ATG5 NM_ SEMA4F NM_ ZDHHC8 NM_ NXF4 ENST HCAP-G NM_ PNPLA2 NM_ FAM76A NM_ RDH5 NM_ FSBP NM_ XPO4 NM_ MTMR10 AL C21orf59 AI C15orf20 AF TBPIP NM_ L3MBTL3 AB TUBA3 NM_ XRCC3 NM_ TFCP2L1 NM_
23 MCM10 NM_ FLJ38608 NM_ FLJ13710 AI GGA2 NM_ FAM62B AB FUT1 NM_ DHX33 AA TRIM6 NM_ PPP2R3B NM_ TNPO1 AL C6orf153 NM_ C2orf7 NM_ HNRPR AK PRKAA1 AI SLC19A1 NM_ C17orf41 NM_ EZH2 NM_ C10orf119 NM_ AK AK DHX37 NM_ MECP2 NM_ LGALS1 NM_ CCNB2 NM_ LOC AL LYPLAL1 NM_ SRGAP2 AB ARHGEF5 NM_ SHMT1 NM_ DDR1 NM_ TACC3 NM_ FLJ27365 AI ECOP NM_ PTTG1IP NM_ RRM2 NM_ DHX33 NM_ PSD3 NM_ COPS7B NM_ CDCA1 NM_ a Each value represents fold reduction for each experimental condition as indicated, as compared to the mock transfection in Hct116 Dicer ex5 cells. Negative values in the luc si transfected cells represent fold increase. b Refseq accession numbers are provided for all annotated genes. mrna accession numbers are provided for those unannotated genes included in our array platform. 23
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