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	Fig. 1. DnaJB6 is a RanGTP-regulated protein associated to the mitotic spindle. (A) Immunofluorescence on HeLa cells transfected with Flag-tagged long and short DnaJB6 isoform-expressing constructs. DnaJB6-L (long) localizes in the nucleus during interphase and to the spindle poles in mitosis. DnaJB6-S (short) shows diffuse cytoplasmic localization both during interphase and mitosis. (B) Western blot analysis of a GST–xDnaJB6-L pulldown experiment in Xenopus laevis egg extract. Importin-β associates with GST–xDnaJB6-L and is released by addition of RanGTP to the extract (top). The lower panel shows that similar levels of GST–xDnaJB6 were used for pull downs in the presence or absence of RanGTP. (C) Amino acid sequence alignment of the putative NLS in human (top) and Xenopus laevis (bottom) DnaJB6 long isoforms. Asterisks highlight identical amino acids. (D) Immunofluorescence images of HeLa cells showing the localization of DnaJB6 in different cell cycle stages using an in-house generated antibody. DnaJB6 accumulates in the nucleus during interphase and localizes to the spindle during mitosis with an accumulation at the spindle poles in metaphase. Tubulin is shown in red, DnaJB6 in green and DNA in blue. Scale bars: 10 μm.
	Fig. 2. DnaJB6 is required for bipolar spindle assembly. (A) Western blot analysis of control and DnaJB6-silenced cell lysates showing the efficiency of DnaJB6 silencing. Cells were lysed 48 h post transfection and 40 μg of protein were loaded per lane. DnaJB6 silencing efficiency was 90% for DnaJB6-L and more than 90% for DnaJB6-S. Tubulin is blotted as protein loading control. (B) Box-and-whisker plot showing the time from nuclear envelope breakdown (NEB) to anaphase onset for control and DnaJB6-silenced cells. Data are from three independent experiments in which a total of 281 control and 250 DanJB6 silenced cells were analyzed. ***P<0.0001 (Mann–Whitney test). (C) Bar graph showing the mean±s.d. distribution of mitotic phases in control (blue) or DnaJB6-silenced HeLa cells (green). Prophase (P), prometaphase (PM), metaphase (M), anaphase (A) and aberrant spindles (Asp) are quantified. Monopolar, multipolar spindles and more disorganized structures were included into the aberrant spindle category (Asp). Data from three independent experiments in which a total of 454 control and 498 DnaJB6-silenced cells were analyzed. *P<0.05, ***P<0.0001 (ANOVA test). (D) Multipolar spindle are highly represented in DnaJB6-silenced cells. Left: representative image of a DnaJB6-silenced HeLa cell with a multipolar spindle. Tubulin is in red and DNA in blue. Scale bar: 10 μm. Right: quantification (mean±s.d.) of the percentage of multipolar spindles in control (blue) and DnaJB6-silenced (green) cells. The percentages are indicated on top of the columns. Data are from three independent experiments in which a total of 765 control and 778 DnaJB6-silenced cells were analyzed. ***P<0.0001 (Fisher's exact test). (E) DnaJB6-silenced cells present extra poles that are mainly not centrosomal. Left: immunofluorescence images of a DnaJB6-silenced HeLa cell with a multipolar spindle showing tubulin (red), centrin (green) and DNA (blue). The arrowhead points to a centrin negative pole. Scale bar: 10 μm. Right: quantification (mean±s.d.) of the percentage of multipolar spindles with poles negative for centrin in control (blue) and DnaJB6-silenced (green) HeLa cells. The percentages are indicated on top of the columns. Data are from two independent experiments in which a total of 126 control and 116 DnaJB6-silenced cells were analyzed. ***P<0.0001 (ANOVA test).
	Fig. 3. DnaJB6 is required for spindle orientation and spindle poles organization. (A) Box-and-whisker plot showing the spindle length in control and DnaJB6-silenced HeLa cells. The spindle length of 267 control and 294 DnaJB6-silenced cells from three independent experiments was measured. A statistically significant increase of the length was detected in DnaJB6-silenced cells. ***P<0.0001 (Mann–Whitney test). (B) DnaJB6-silenced cells have ectopic microtubule clusters in metaphase. Left: immunofluorescence images of a representative DnaJB6-silenced metaphase cell with an ectopic microtubule cluster (white arrowhead). Tubulin is shown in red and DNA in blue. Scale bar: 10 μm. Right: bar graph showing the percentage of bipolar spindles containing ectopic microtubule clusters in control and DnaJB6-silenced HeLa cells. 182 control and 192 DnaJB6-silenced cells in metaphase were examined. ***P<0.001 (Fisher's exact test). (C) DnaJB6-silenced cells present spindle orientation defects. Left: representative images of a control cell with a correctly oriented spindle in which the two centrosomes are aligned with the substrate (top image) and a DnaJB6-silenced cell with a misoriented spindle (bottom image; in this case, only one centrosome is visible due to the location of the two poles on two different focal planes). Tubulin is in red, centrin in green and DNA in blue. Scale bar: 10 μm. Right: quantification of the mean±s.d. percentage of metaphase HeLa cells that present misoriented spindles. Spindles were scored as not correctly oriented with the substrate when the two centrosomes were not on the same focal plane. Anti-centrin antibodies were used to determine centrosome position. The percentages are indicated on top of the columns. Data from four independent experiments in which a total of 600 cells were analyzed for condition. ***P<0.001 (Student's t-test). (D) DnaJB6-silenced cells show severe spindle pole focusing defects. Left: immunofluorescence images of control and DnaJB6-silenced HeLa cells showing tubulin (red), centrin (green) and DNA (blue). Spindles from a control and a DnaJB6-silenced are shown as examples of focused and open spindle poles, respectively. Double arrows exemplify poles width measurement. Scale bar: 10 μm. Right: box-and-whisker plot showing measures of spindle pole width in control and DnaJB6-silenced HeLa cells. Bipolar metaphase spindles with correctly aligned chromosomes were randomly selected. The tubulin signal was thresholded and a line was drawn crossing the centrosome (centrin staining) and connecting the two closest spindle borders. Graph shows the results of a representative experiment out of two in which a total of 90 poles were measured in each condition. ***P<0.001 (Student's t-test). (E) DnaJB6-silenced cells show centrosome mispositioning relative to the spindle pole position. Left: representative images of a control and two DnaJB6-silenced cells. Centrosomes are laterally displaced or detached from the spindle poles in DnaJB6-silenced cells. Tubulin is in red, centrin is green and DNA is blue. Scale bar: 10 μm. Right: quantification of the centrosome position defects. When the centrosomes move laterally the centrosome axis is deviated. The left-hand box-and-whisker plot represents the centrosome axis deviation in control and DnaJB6-silenced cells. This deviation is calculated by measuring the α and subtracting this from 90, where α is the angle between the centrosome axis and the metaphase plate, as shown in the schematic representation. When the centrosomes are detached from the poles a midpoint is fixed (yellow cross) and distance from the metaphase plate (h) measured and plotted (shown on the right). The plots show data from a representative experiment where more than 30 cells were analyzed for each condition. ***P<0.001 (Student's t-test).
	Fig. 4. DnaJB6 is involved in spindle microtubules organization. (A) Immunofluorescence images of mitotic control and DnaJB6-silenced HeLa cells undergoing microtubule regrowth after nocodazole washout. Cells were fixed at the times indicated after nocodazole washout and processed for immunofluorescence to visualize the microtubules (tubulin, shown in gray) and DNA (not shown). The white arrowheads point to microtubule asters that fail to incorporate into the main spindle poles. Scale bar: 10 μm. (B) Quantification of the percentage of cells with two asters at different time points after nocodazole (NOC) washout as shown in A. The blue line corresponds to the control cells and the green line to DnaJB6-silenced cells. Data are from three independent experiments in which more than 300 cells were recorded for each time point. ***P<0.001 (Fisher's exact test). (C) Representative immunofluorescence image of a HeLa cell fixed at 60 min after nocodazole washout with two microtubule asters that do (top) or do not (bottom) organize a bipolar spindle. Tubulin is in red, and DNA in blue. Scale bar: 10 μm. Quantification of the mean±s.d. percentage of cells having two microtubule asters organized into a bipolar spindle 60 min after nocodazole washout. Data from two independent experiments in which 503 control (blue) and 638 DnaJB6-silenced cells (green) were analyzed. The exact percentages are indicated on top of each bar. ***P<0.001 (Fisher's exact test).
	Fig. 5. DnaJB6 depletion from Xenopus egg extracts induces spindle pole organization defects. (A) Western blot analysis of control and xDnaJB6-depleted (Δ-DnaJB6) Xenopus egg extracts (EE) without (−) or with (+) addition of the recombinant MBP–xDnaJB6-L. The depletion was very efficient and the recombinant protein was added at close to endogenous concentrations. 1 μl of egg extract was loaded per lane. The endogenous and recombinant proteins were detected with an in-house generated anti-xDnaJB6 antibody. (B) Fluorescence images of spindles assembled in control or DnaJB6-depleted egg extracts. Most spindles assembled in control-depleted extracts have focused spindle poles, whereas many spindles assembled in DnaJB6-depleted extracts show spindle pole focusing defects defined as ‘Split poles’ or ‘Open poles’ as shown. The white arrowheads point to the different types of spindle poles. In the merge, tubulin is shown in red and DNA in blue. Scale bar: 10 μm. (C) Graph showing the mean±s.d. percentage of focused spindle poles in control extracts (blue), DnaJB6-depleted extracts (green) and DnaJB6-depleted extracts containing MBP–xDnaJB6 (orange). Data are from four independent experiments in which 703, 607 and 358 spindle poles were examined, respectively. ***P<0.001 (ANOVA test). (D) Fluorescence image of a spindle assembled in egg extract supplemented with an excess of DnaJB6 (1 μM) showing tightly focused spindle poles (white arrowheads). Scale bar: 10 μm. Bar graph on the right shows the percentage of tightly focused poles in spindles assembled in control extracts supplemented with MBP (blue) and in extracts supplemented with MBP–xDnaJB6 (purple). Data are from three independent experiments in which 114 and 258 spindle poles were analyzed. ***P<0.0001 (Fisher exact test).
	Fig. 6. DnaJB6 regulates dynactin spindle localization and is required for dynein-dependent force generation within the spindle. (A) Western blot analysis of a MBP–xDnaJB6-L pulldown experiment in Xenopus egg extract. MBP–xDnaJB6-L- or MBP-coated Dynabeads were incubated in egg extracts in the presence or absence of RanGTP. p150Glued was specifically pulled down with MBP–xDnaJB6-L from extracts containing RanGTP. The lower panel shows that similar amounts of MBP–xDnaJB6-L was used for pulldown both in the presence and absence of RanGTP. (B) Western blot analysis showing the position of p150Glued in 8–20% sucrose density gradients from lysates of control and DnaJB6-silenced cells. Lysates were prepared from mitotic cells. KI was added to the lysates and incubated for 1 h before running the gradients, at the concentrations indicated on the right. A shift to in where p150Glued is detected in to fractions with a lower percentage of sucrose is observed in mitotic DnaJB6 cell lysates treated with 150 mM KI compared to the mitotic control cells lysates with the same treatment, as highlighted with asterisks. (C) p150Glued accumulates at spindle poles in DnaJB6-silenced cells. Left: representative immunofluorescence images from control and DnaJB6-silenced HeLa cells showing the localization of p150Glued in metaphase spindles. In the merge, tubulin is in red, dynein is in green and DNA is in blue. Scale bar: 10 μm. Right: box-and-whisker plot showing the value of the of p150Glued signal normalized to that of the tubulin signal (a.u., arbitrary units) in each spindle pole in control and DnaJB6-silenced HeLa cells. More than 30 cells were analyzed for each condition in one representative out of three independent experiments. (D) DnaJB6 silencing rescues spindle bipolarity in STLC-treated HeLa cells. Left: immunofluorescence images of representative monopolar and bipolar spindles in DnaJB6-silenced HeLa cells incubated with STLC. Tubulin is shown in green and DNA in blue. Scale bar: 10 μm. Right: bars graph showing the mean±s.d. percentage of bipolar spindles in control or DnaJB6-silenced HeLa cells incubated with STLC. Data from three independent experiments in which 937 control and 1057 DnaJB6-silenced cells were analyzed. ***P<0.001 (ANOVA test).

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