Supplemental Table 1. Primers and Probes for RT-Pcrs

Home , E2F2, E2F3, GABPA, Sp1 transcription factor

Supplemental Table 1. Primers and probes for RT-PCRs

Gene Direction Sequence

Quantitative RT-PCR E2F1 Forward Primer 5’-GGA TTT CAC ACC TTT TCC TGG AT-3’ Reverse Primer 5’-CCT GGA AAC TGA CCA TCA GTA CCT-3’ Probe 5’-FAM-CGA GCT GGC CCA CTG CTC TCG-TAMRA-3' E2F2 Forward Primer 5'-TCC CAA TCC CCT CCA GAT C-3' Reverse Primer 5'-CAA GTT GTG CGA TGC CTG C-3' Probe 5' -FAM-TCC TTT TGG CCG GCA GCC G-TAMRA-3' E2F3a Forward Primer 5’-TTT AAA CCA TCT GAG AGG TAC TGA TGA-3’ Reverse Primer 5’-CGG CCC TCC GGC AA-3’ Probe 5’-FAM-CGC TTT CTC CTA GCT CCA GCC TTC G-TAMRA-3’ E2F3b Forward Primer 5’-TTT AAA CCA TCT GAG AGG TAC TGA TGA-3’ Reverse Primer 5’-CCC TTA CAG CAG CAG GCA A-3’ Probe 5’-FAM-CGC TTT CTC CTA GCT CCA GCC TTC G-TAMRA-3’ IRF-1 Forward Primer 5’-TTT GTA TCG GCC TGT GTG AAT G-3’ Reverse Primer 5’-AAG CAT GGC TGG GAC ATC A-3’ Probe 5’-FAM-CAG CTC CGG AAC AAA CAG GCA TCC TT-TAMRA-3' IRF-2 Forward Primer 5'-CGC CCC TCG GCA CTC T-3' Reverse Primer 5'-TCT TCC TAT GCA GAA AGC GAA AC-3' Probe 5'-FAM-TTC ATC GCT GGG CAC ACT ATC AGT-TAMRA-3' TBP Forward Primer 5’-CAC GAA CCA CGG CAC TGA TT-3’ Reverse Primer 5’-TTT TCT TGC TGC CAG TCT GGA C-3’ Probe 5’-FAM-TGT GCA CAG GAG CCA AGA GTG AAG A-BHQ1-3’

Primers and Probes for quantitative RT-PCRs were designed using the computer program “Primer Express” (Applied Biosystems, Foster City, CA, USA).

1 Supplemental Table 2. Sequences of siRNA oligonucleotides

Gene Sequence

E2F3a #1 5´-AAG GGC ACU GCU AGC CAG CTT-3´ E2F3a #2 5´-CAC UUC CAC CAC CUC CUG UTT-3´

IRF-1 5´-CCA AGA ACC AGA GAA AAG ATT-3´

IRF-2 5´-CUC UUU AGA AAC UGG GCA ATT-3´

Scrambled 5´-UAA UGU AUU GGA ACG CAU ATT-3´

All siRNA oligonucleotides were purcased from Eurofins MWG (Eurofins MWG GmbH, Ebersberg, Germany).

2 Supplemental Table 3. Transcription factor binding site profiles

Symbol Official Full Name Accession E2F3a E2F3b E2F1 E2F2 -2018 to orf Intron 1/2 -2015 to orf -2065 to orf

CREB1 cAMP reponse element-binding protein P16220 + + + + E2F1 E2F transcription factor 1 NP005216 + + + + ELK1 ETS oncogene family P19419 + + + + ELK 4 ETS oncogene family P28324 + + + + FOXC1 Forkhead box protein C1 Q12948 + + + + FOXD1 Forkhead box protein D1 Q16676 + + + FOXI1 Forkhead box protein I1 Q14518 + + + + FOXL1 Forkhead box protein L1 Q12952 + + + + GABPA GA-binding protein alpha chain Q06546 + + + + GATA2 GATA binding protein 2 P23769 + + + + GATA3 GATA binding protein 3 P23771 + + + + HLF hepatic leukaemia factor Q16534 + + + + IRF-1 Interferon regulatory factor-1 P10914 + + + + IRF-2 Interferon regulatory factor-2 P14316 + MAX MYC associated factor X AAH36092 + + + MEF2A myocyte enhancer factor 2A EAX02249 + + + + MYC c-Myc NM002458 + + + NFIL3 nuclear factor, interleukin 3 regulated NP005375 + + + + NR2F1 nuclear receptor subfamily, F1 P10589 + + + + REL c-REL, member of Rel/NFKB family Q04864 + + + + RELA reticuloendotheliosis viral oncogene A Q04206 + + + + RORA RAR-related orphan receptor A NP599023 + + + + RUNX1 Run-related transcription factor 1 AAI44054 + + + + RXR-VDR Vitamin D3 receptor P11473 + SOX9 SRY [Sex determining region Y]-box 9 P48436 + + + + SP1 SP1 transcription factor P08047 + + + + SPI1 SFFV proviral integration oncogene spi1 P17947 + + + + SPIB Spi-B transcription factor Q019892 + + + + SRF Serum response factor P11831 + SRY Sex determining region Y Q05066 + + + + TAL1-TCF3 T-cell acute lymphocytic leukemia protein 1 P17542 + + + + TEAD SV40 transcriptional enhancer factor P28347 + + + + TFAP2A Transcription factor AP-2 alpha P05549 + + + + USF1 upstream transcription factor 1 P22415 + + + + YY1 YY1 transcription factor P25490 + + + + ZNF42 MZF 1 (myeloid zinc finger 1) AAH53316 + + + +

36 different putative transcription factor binding sites (TFBS) were estimated in 5’-UTRs of E2F1, E2F2, E2F3a and E2F3b. Overall homogeneity between proliferation-promoting E2Fs was 92%. orf, open reading frame.

3 Figure legends to Supplementary Figures

Supplemental Figure 1. A-B, OVCAR-3 and MDAH-2774 cells under EGF exposure. EGF treatment yielded a selective, time-dependent up-regulation of E2F3a mRNA (white bars), whereas

E2F3b (black bars) was not inducible. *, p < 0.05, **, p < 0.01. Bars indicate the mean ± SD of three independent experiments. C, EGF mediated growth enhancement of OVCAR-3 and MDA-2774.

Asterisks indicate significant difference in cell proliferation compared to untreated controls. D,

Constitutive expression levels of EGFR protein in ovarian cancer cell lines, the mesothelioma cell line CRL-5820 and in healthy HPMC and OSE. The arrow indicates EGFR immunoreactivity at 170 kDa. Shown is a representative blot of three independent experiments.

Supplemental Figure 2. DNA sequences of the 5’-flanking regions of the human E2F1, E2F2 and

E2F3 genes. In extension to the published sequences of the E2F1, E2F2 and E2F3 promoters

(21,22,13) we included the first 2000bp upstream the open reading frame in our promoter analyses.

As shown for the mouse E2F3 gene we assumed the putative E2F3b promoter region to be located in intron 1 of the E2F3 gene (13). Boxes indicate putative c-myc, IRF-1, IRF-2 and E2F binding sites.

Promoter regions of all investigated E2F transcription factors contain IRF-1 recognition sites, whereas IRF-2 is selectively located in the 5’-UTR of the E2F3a gene.

Supplemental Figure 3. A, Changes in E2F3b expression levels under IFNγ exposure. HOC-7,

HTB-77 and A2780 were incubated with 10 ng/ml IFNγ over various time periods, and quantitative expression of E2F3b mRNA was determined with RT-PCR and normalized to TBP expression. Bars indicate E2F3b mRNA levels referred to expression levels of untreated controls (Co). *, p<0.05. B,

Coincubation of ovarian cancer cells with EGF and IFNγ did not yield any change in E2F3b transcript levels. *, p<0.05. C, Basal expression levels of E2F3a and E2F3b mRNA levels in various ovarian cancer cell lines, the mesothelioma cell line CRL5820, HPMC and OSE normalized to TBP expression. Note the low E2F3 isoform transcript levels in SKOV-6 indicated by an arrow.