Fig 2: Distribution of NPM1 by simple cell fractionation. (A) Summary of the process of cell fractionation. (B) Distribution of NPM1 in the sample prepared in A. (C) Immunofluorescence of endogenous NPM1. (Upper panel) HeLa cells were fixed with formaldehyde, then permeabilized using Triton X-100. (Lower panel) HeLa cells were permeabilized using Triton X-100, then fixed with formaldehyde. Scale bar, 10 μm.

Fig 3: Microtubule dynamics analysis using the EB3-tagRFP plus-end tracking protein.(A) Still frames from time-lapse experiments show EB3-tagRFP signals. Upper left image represents EB3-tagRFP signal at t = 0s. On the right of the image, enlarged images of an EB3-tagRFP comet evolution (displayed on the full size image) are shown (upper enlarged panels from t = 0s to t = 19.2s). The plusTipTracker software package detects and tracks all the microtubule growing ends as exemplified on the enlarged images (lower enlarged panels). Lower image represents a temporal color code image of the EB3-tagRFP signal evolution from t = 0s to t = 19.2s. The color code scale is presented on the right. Scale bars represent 10µm. (B) Schematic representation of EB3-tagRFP tracking using the plusTipTracker software. Microtubule growing ends are schematized with the red circles. Transparent red circles represent temporarily comet disappearance. After comet detection and tracking, the software classifies the sub-tracks in three groups: polymerization, pause and catastrophe. For more details see [25][27–30]. (C) Inhibition of nucleolin or B23 expression by siRNA. Western-blot analysis of whole cell extracts from untransfected control, mock transfected, control siRNA, nucleolin siRNA (siRNA concentration 2nM or 20nM) and B23 siRNA transfected cells. Cells were harvested four days after transfection and protein extracts were analyzed by western-blot using a nucleolin polyclonal antibody (detected with a secondary antibody coupled to IRdye800) and B23, a-tubulin and ß-actin monoclonal antibodies (detected with secondary antibody coupled to Alexa680). (D) Quantifications of the fluorescent western blots, displayed in (C), expressed as a ratio of nucleolin over ß-actin (left) or B23 over ß-actin (right). The normalized expression level of nucleolin or B23 proteins was set to 100% in control cells (NCL siRNA 22.60% and B23 siRNA 20.59%). (E) Nucleolin over expression by transient transfection of a NCL-GFP construct. On the upper left image, the GFP channel is presented [black and white]. Arrows show nucleoli (nucleolin-GFP signal) and the centrosomes (C1G: Centrin-1-GFP signal) and cell outlines are shown. Lower image represents a temporal color code image of the EB3-tagRFP signal evolution for 40s. The color code scale is presented on the right of the image. Upper right image merge image of the GFP channel and the temporal color code image of the EB3-tagRFP signal is presented. Scale bars represent 10µm.

Fig 4: Nucleolin expression affects microtubule catastrophes.Analysis of catastrophe events in control [black], nucleolin depleted [dark red], nucleolin over expressing [dark blue], B23 depleted [light red] and B23 over expressing cells [light blue]. The asterisks indicate that the mean value differs from the control condition with the Wilcoxon-Mann-Whitney two-sample rank test at different significance levels: 10% (* P<0.1), 5% (** P<0.05), 0,5% (*** P<0.005). (A) Catastrophe frequency (10-3s-1) is calculated by dividing the time microtubules spend in growth (B)-(D) Catastrophe parameters are deduced by linking two temporally adjacent growths spatially spaced out by a Bgap (Backward Gap). The mean values of speed (B), life time (C) and length (D) during catastrophes were measured in each cell and reported on the graphs. (E) and (F): 2D graphs showing the repartition of the cells according to depolymerization speed (x-axis) and length (y-axis). Control [black], nucleolin over expressing [dark blue] and nucleolin depleted [dark red] (E) or B23 over expressing [light blue] and B23 depleted [light red] (F) cells are represented by the squares. (A-D) Cells were individually analyzed (control: 17 cells; NCL-siRNA: 24 cells; NCL-GFP: 10 cells; B23-siRNA: 11 cells; B23-GFP: 14 cells)

Fig 5: Subnuclear distribution of GFP fused to deletion or substitution mutants of Ilf3/NF90 NoLS.(A) Plasmids pEGFP-N1 (Control), pEGFP-N1-Ilf3/NF90 common N-terminal long region (NoLS-GFP), pEGFP-N1-Ilf3/NF90 common N-terminal short region (N-GFP), pEGP-N1/deletion mutant of the four-arginine stretch from the NoLS (?4R-GFP), pEGP-N1/substitution mutant of the threonine 10 by alanine (T10A-GFP) and pEGP-N1/substitution mutant of the threonine 10 by aspartate (T10D-GFP) and (B) plasmids pEGP-N1/deletion mutant of the three-histidine stretch (?3H-GFP), pEGP-N1/substitution mutant of the three-histidine stretch by three alanines (3H->3A-GFP), three phenylalanines (3H->3F-GFP), three lysines (3H->3K-GFP) or three glutamates (3H->3E-GFP) and pEGP-N1/substitution mutant of the three-histidine stretch by three lysines/deletion mutant of the four-arginine stretch (3H->3K/?4R-GFP) were transfected into HeLa cells. After 24 hours, cells were co-stained with the monoclonal anti-B23 antibody (B23) and DAPI. After confocal microscopy acquisition, focal planes exhibiting a B23 optimal signal were chosen. GFP fusion proteins appear in green, B23 in red and DAPI in blue.