Supplementary Information

Fig. S1. Ultrastructural patterns of the “nucleolar disruption”.

(a) U2OS cell after 4 h exposure to 8 nM Actinomycin D. The ribonucleoprotein structures of the nucleolus appears to be segregated into three main components: a light fibrillar, a dense fibrillar and a granular component. Uranium and lead staining. (b) Isolated rat hepatocyte treated with α-amanitin at the dose of 5μg/ml for 3 h. The nucleolar body appears to be disrupted into three fragments in which the ribonucleoprotein components are segregated. Uranium and lead staining. Scale bar, 0.5 mm.

Fig. S2. 5-Fluorouracil exposure inhibits the synthesis of rRNA without inducing the “nucleolar disruption”.

(a) Representative Western blot of p53 expression in U2OS cell exposed to 75 μM 5-fluorouracil for 4 h. Ctrl, control cells; +5FU, cells treated with the drug. (b) Quantitative analysis of 28S and 18S rRNA of control and 5FU- treated U2OS cells. RNA was size-separated on 1% agarose gel and stained with ethidium bromide. Two bands corresponding to 28S and 18S rRNA were visible in each lane. Histograms show the values (mean ± s.d.) of three experiments. (c) Nucleolar fine structural organisation of a U2OS cell, treated with 5FU. Arrows indicate the fibrillar centres, surrounded by the ribonucleoprotein fibrillar and granular component. Uranium and lead staining. Scale bar, 0.4 mm. (d) Visualisation of nucleophosmin distribution in U2OS cells treated with 5-FU: 1) DAPI staining, 2) nucleophosmin labelling with monoclonal antibodies versus nucleophosmin, revealed by FITCH-conjugated secondary antibodies 3) merging of image 1 and 2. Scale bar, 10 mm.

Fig. S3. POLR1A interference does not modify the distribution of MDM2 in the U2OS cell nuclei.

Distribution of MDM2 in control and in POLR1A-silenced U2OS cells, 48 h after the end of the silencing procedure, as visualised by anti-MDM2 rabbit polyclonal antibody immunostaining revealed by FITCH-conjugated secondary antibodies. Scale bar, 10 mm.

Fig. S4. Actinomycin D treatment abolishes rRNA transcription in U2OS serum-starved cells.

Serum-starved U2OS cells were exposed to 8 nM Actinomycin D for 4 h. 1) DAPI staining reveals unstained round structures corresponding to nucleolar bodies. 2) 5-fluorouridine immunofluorescent staining is uniformly distributed through the nucleoplasm, but not in the nucleolar bodies which are unlabelled. 3) Merging of image 1 and 2. Scale bar, 10 mm.

Fig. S5. Short-term Gemcitabine treatment does not modify the synthesis of rRNA in MCF-7 cells.

MCF-7 cells were treated with 10 mg/ml Gemcitabine for 4 hours. The synthesis of rRNA was evaluated by real time RT-PCR.