Legends Supplementary Figure 1. GST pull-down analysis of the interaction of GST- (A, B), GST mutants (B) or GST- (C) with indicated proteins. A, B, Cell lysate from untransfected HeLa cells were loaded on GST, GST- or GST- mutants immobilized on glutathione sepharose beads. Interactions were evaluated by immunoblotting. C. Cell lysate from HeLa cells transfected with indicated FLAG-constructs was incubated with GST or GST- immobilized on glutathione sepharose beads. Interactions were evaluated by immunoblotting. D. Endogenous was immunoprecipitated from the cytoplasm (cyto) and nucleus (nuclei)-enriched fractions with anti- or irrelevant goat Ig (IgG) before immunoblot analysis of and. MW: molecular weight. E. GST pull-down analysis of the competitive interaction of with or. GST- immobilized on glutathione sepharose beads was incubated in the presence of a constant amount of cells lysate from -transfected 293T cells and increasing amount of cell lysate from -transfected 293T cells. Interactions were evaluated by or immunoblotting. Supplementary Figure 2. Gene luciferase experiments performed in HeLa (A) HT-29 (B) and U2-OS (C) cells transfected with CCNE promoter-luciferase reporter plasmid (pccne), along with indicated constructs. Luciferase activity was normalized to β-galactosidase activity and expressed as fold induction of promoter stimulated by empty vector alone. Mean ± sd of one representative experiment. Upper panels: the efficiency of expression is checked by immunoblotting. Supplementary Figure 3. Quantitative RT-PCR analysis of ccne or birc2 mrna in HT-29 cells transfected with empty or encoding vectors. Results normalized to hprt mrna and expressed relative to empty vector. Mean ± SD of one representative experiment. Supplementary Figure 4. Flow cytometry analysis of cell cycle repartition in HeLa cell synchronized in late G1 phase by a thymidine double block. Cell cycle is evaluated in asynchronized (As) and in
synchronized cells studied, 2, 4, 6 and 8 hours after block release by flow cytometry after BrdU and PI staining of cells. Supplementary Figure 5. Chromatin immunoprecipitation experiments performed using an anti-, anti- or an irrelevant antibody (Ig) in HeLa (A) or CaSki (B) cells. The genomic DNA region encompassing one E2F-binding site of the CCNE ( or CCNA (p(ccna) promoters were amplified by PCR. A, HeLa cell were synchronized in late G1 phase by a thymidine double block and ChIP is performed, 2, 4, 6 and 8 hours after block release. B, CaSki cells were transfected with control (si-co) or -targetted si-rna (si-) 48 hours before ChIP analysis. Supplementary Figure 6. A, Quantitative RT-PCR analysis of CCNE mrna in MEF form wild type (wt) or -deleted mice ( -/- ). Results normalized to HPRT mrna and expressed relative to MEF wt. Mean ± SD of one representative experiment. B, Immunoblot analysis of, cyclin E, cyclin A in MEF form wild type (wt) or -deleted mice ( -/- ). HSC7: loading control. C, MEF were stained with Hoechst 33 352 (7.5µg/ml, 1 hour) and then sorted in function of their DNA content. Upper, Flow cytometry analysis of DNA content before (As) and after sorting. Lower, Immunoblot analysis of, XIAP and in the cytoplasm (C) and nucleus (N)-enriched fractions of sorted MEF cells. HSC7: loading control. D, Gene luciferase experiments performed in MEF -/- cells transfected with CCNE promoter-luciferase reporter plasmids, along with control (Co) or and/or -encoding vector. Luciferase activity was normalized to β-galactosidase activity and expressed as fold induction of promoter stimulated by empty vector alone. Mean ± sd of one representative experiment.
Supplementary Figure 1 A B Rb p53 - - 6 kda 11 kda 53 kda GST 6 kda kda 96 72 55 43 PCNA - 36 kda 34 26 Cyclin A Cyclin B chk1 p65 p21 TTK Rad52 IB GST - 55 kda 62 kda 65 kda 21 kda 97 kda 4 kda 98 kda C FLAG- Input FLAG-BIR1-3 FLAG-CARD-RING FLAG- GST 72 FLAG-BIR1-3 FLAG-CARD-RING FLAG- GST- FLAG-BIR1-3 FLAG-CARD-RING kda 55 43 34 26 26 kda GST 86 26 D Input (1/1) IP IgG MW Input (1/1) IP IgG E GST GST- : - : 6 kda Pull-down Cyto. Nuclei GST- 98 kda GST Input 26 kda 6 kda
Supplementary Figure 2 A HSC7 Vector L47A Fold induction/vector 3 25 2 15 1 5 Vector B HT-29 C U2-OS HSC7 Vector Vector HSC7 Fold induction/vector 7 6 5 4 3 2 1 16 14 12 1 8 6 4 2
Supplementary Figure 3 Fold induction/ vector (AU) 14 birc2 12 1 8 6 4 2 2,5 2 1,5 1,5 ccne
Supplementary Figure 4 As 2 4 8 BrDu Number of cells Propidium iodide
Supplementary Figure 5 A ChIP ChIP Input (1/1) ChIP Ig 2 4 8 Input (1/1) ChIP Ig 2 4 8 p(ccna) B si-co Input Ig si-
Supplementary Figure 6 A B mrna relative expression 2. ccne mrna 1.5 1..5. MEF: wt -/- Cyclin E 5 kda Cyclin A 55 kda HSC7 MEF: wt -/- C As G G 1 S G 2 M XIAP HSC7 C N C N C N 6 kda D Fold induction/co 12 1 8 6 4 2 vector Co