b

c80

1 2 3 4 50

10

20

40

120

ratio

(N

/C)

5

15

d

Fig. S1 Nuclear localization of NtE2F is assumed by its N terminal NLS. (a) Various constructs were transitory expressed in mid-log phase BY-2 cells using GFP fused either to NtE2F or : to NLS in frame with the cytoplasmic Chalcone synthase cloned in the PCK vector (Guerra-Peraza et al., 2005). The following constructs were analyzed PCK (1), PCK:NLS (2), PCK:NLSMU (3), GFP:NtE2FMUNLS (4), GFP:NtE2F (5) and GFP as a control. (b) sequence of E2F-NLS and its mutation are indicated. (c) Nuclear/cytoplasm signal ratio was evaluated for the various constructs (1-5) with the ImageJ softmare. S.D. are indicated. Bars represent 10 µm. Images were captured on confocal microscopy. (d) E2F factors are mainly present in the proteic nuclear fraction. Cellular fractionation was performed on mid-log BY-2 cells as described in the supplementary file material and methods. Western analysis of (1) 10 µg of proteins from the cytosolic (1) or nuclear (2) fraction were performed with either an antibody anti-PCNA as a marker of the nuclear fraction or an antibody anti Human E2F previously used to reveal E2F factors in tobacco (Chabouté et al., 2000). As a loading control (LC), Coomassie blue staining of the immunoblot is presented.

Chabouté ME, Clément B, Sekine M, Philipps G, Chaubet-Gigot N. 2000. Cell cycle regulation of the tobacco ribonucleotide reductase small subunit gene is mediated by E2F-like elements. Plant Cell 12: 1987-2000.Guerra-Peraza O, Kirk D, Seltzer V, Veluthambi K, Schmit AC, Hohn T, Herzog E. 2005. Coat proteins of Rice tungro bacilliform virus and Mungbean yellow mosaic virus contain multiple nuclear-localization signals and interact with importin alpha. J Gen Virol 86: 1815–1826Uemukai K, Iwakawa H, Kosugi S, de Uemukai S, Kato K, Kondorosi E, Murray JA, Ito M, Shinmyo A, Sekine M. 2005. Transcriptional activation of tobacco E2F is repressed by co-transfection with the retinoblastoma-related protein: cyclin D expression overcomes this repressor activity. Plant Mol Biol 57: 83-100

E2F-NLS: PLKRKSE

E2F-NLS Mu: PLAAASE

anti-PCNA

LC

CC N1 2

anti-humanE2F

PCK (1) PCK:NLSMU (3)PCK:NLS (2)

GFP-NTE2F

a

GFPGFP:NtE2F (5)GFP:NtE2FNLSMU (4)

kDa

66

45

Supporting Information Figs S1-S3

a

b

Fig. S2 The GFP:NtE2F fusion is functional. (a) Mid-log BY-2 cells expressing the Luciferase reporter gene under the control of NtRNR1b promoter were transfected either with empty vector (control) or GFP-NtE2F plus DP-RFP vectors. Cells were selected for their fluorescence signals 5 hours after cell transfection and luciferase activity was evaluated as previously described (Chabouté et al,2000). SDs are indicated. (b) GFP:NtE2F can rescue the TSO2 down regulation observed in e2fa (Roa et al., 2009) in response to BLM. Non-treated plants were used as a control. 18Swas used as a standard. Experiments were performed by RT-qPCR.

control NtE2F + DP

WT e2fa NtE2F x e2fa

Contrôle100

10070

70

pH

pH

4.0 7.0

4.0 7.0

Fig. S3 Post-translational modification of GFP:NtE2F in BY-2 cells treated with BLMNuclear proteic extracts (75 g) were prepared from mid-log BY-2 cells expressing the GFP:NtE2F fusion treated with BLM (10-5M, 1.5h) (b) and non-treated as a control (a). Samples were run on 2 DE. Immobilized, pH 4–7 gradient strips (Readystrip IPG, Biorad) were run on the Proteon IEF Cell (Biorad) for the first dimension. The second dimension was run on a 12% SDS-PAGE gel. The gel was stained with colloidal Coomassie and western blot was performed using an antibody antiGFP. Enlargements of the spot containing GFP:NtE2F are presented. The presence of additional spots after BLM treatment are correlated to multiple post-translational modifications.

pH4.0 7.0

pH4.0 7.0

kDa

kDa

Control

BLM

a

b