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	Figure 1. A screen to identify NETs that recruit chromosomes to the nuclear periphery. (a) An integrated lacO array marks chromosome 5 that tends to not be at the periphery in line 5.1. NETs fused to mRFP were exogenously expressed to screen for those involved in tethering chromatin to the NE. (b) Detection of the lacO array (arrowheads) and the transfected NETs (red) in the 5.1 line. The lacO array was not observed at the periphery in the untransfected cells (left) or the NET55 transfected cells, but was observed at the periphery in NET29 and NET39 transfected cells. Scale bar, 5 μm. (c) lacO array position was determined relative to five shells of equal area eroded from the periphery (1) to the centre (5) of the nucleus. To avoid errors in z from lacO arrays at the top or bottom of the nucleus, cells were only analyzed if the array occurred in the midplane where arrays occurring in the inner shells would be clearly internal. (d) Percentage of lacO-tagged loci in each of five erosion shells is plotted with the nuclear periphery (shell 1) to the left in dark blue and more internal localization occurring to the right and with lightening shades of blue. n = 50 cells for each NET. Single asterisks designate NETs with lower stringency P-values < 0.05 comparing the position of the array in the NET-transfected cells to the mRFP control using χ2 tests; double asterisks designate NETs with higher stringency P-values < 0.01. Statistics for all NETs are given in Additional file 1. UT, untransfected.
	Figure 2. NETs have distinct effects on a lacO array inserted into chromosome 13. (a) In a different cell line the lacO array inserted in chromosome 13q tends to be at the periphery: so a NET could promote more peripheral localization or less. (b) Percentage of lacO-tagged 13q loci in each of five erosion shells is plotted with the nuclear periphery (shell 1) to the left in dark blue and more internal localization occurring to the right and with lightening shades of blue. n = 50 cells for each NET. (c) NETs that had strong effects on either cell line and two controls that did not (NET37 and NET55) were retested in two additional experiments to generate standard deviations and P-values. *P < 0.05 and **P < 0.01 comparing the position of the array in the NET-transfected cells to the mRFP control using χ2 tests. Effects of individual NETs were highly repeatable with small error bars for standard deviation from the mean and P-values < 0.0001 for NET5, NET29, NET39, NET45 and NET47 in line 5.1. Statistics for each NET are given in Additional file 1. UT, untransfected.
	Figure 3. Positioning effects are not due to changes in nuclear size and shape, distribution of other nuclear proteins, or loss of nuclear permeability. HT1080 cells transiently transfected with tagged NETs were tested for parameters that could theoretically indirectly influence chromosome position. (a) The area of the nuclear midplane (left, distribution plots) and the longest and shortest distance across it (right, scatter plots) were measured and quantified for over 40 transfected cells for each NET. In each case the NET transfected cells were compared to untransfected (UT) cells in the same population. Only very moderate fluctuations in nuclear size or shape were observed. (b) Transfected cells were stained with antibodies for nucleolin to determine if NETs had indirect disruptive effects on nucleoli and for lamins and the NET emerin to determine if NETs generally altered NE organization. No changes in the distribution of these markers were observed. (c) The permeability barrier function of the NE in NET-transfected HT1080 cells was tested by expressing a reporter transport cargo in the same cells. The cargo contains two GFP molecules in tandem with a strong nuclear localization signal and a weak nuclear export signal so that it can shuttle but accumulates predominantly in the nucleus. If nucleocytoplasmic transport or NE permeability is compromised, the reporter accumulates more in the cytoplasm as when the herpesvirus protein ICP27, which interferes with nuclear transport through binding to Nup62, is co-expressed (second panel). None of the NETs affected the distribution of the reporter. For both (b) and (c) scale bar = 10 μm.
	Figure 4. NETs promote movement of whole chromosomes independently of the lacO array. (a) NET-transfected cells subjected to FISH with whole chromosome paints and a probe for lacO. For NET39 both copies of chromosome 5 were in direct proximity to the NE, whereas the singly integrated array was merely closer; thus, relocalization to the periphery is independent of lacO sequences. (b) FISH with chromosome 5 paint on NET transfected HT1080 cells lacking a lacO array validated the lacO screen results. (c) Average DAPI fluorescence intensity in each nuclear erosion shell. No differences in DAPI distribution could be observed between the control cells and the NET transfected cells. n = 50 cells for each NET. (d) Quantification of chromosome 19 pixel intensity in the shells generated by the erosion macro. For the graph the percentage of the total intensity in the two outermost shells is summed (1+2, peripheral) and plotted against the summed percentage of the total intensity in the two innermost shells (4+5, internal). Error bars indicate standard deviation between the means of individual experiments. For both (a) and (b) scale bar = 5 μm.
	Figure 5. NETs promote movement of partly overlapping and partly distinct sets of chromosomes. (a) Similar to the two-dimensional analysis, an erosion approach was used to divide the three-dimensional nucleus (three-dimensional reconstructions) into shells of equal volume, but six shells instead of the five for the two-dimensional analysis were generated. (b) Quantification of whole chromosome painting in the absence of the array. Mean (± standard deviation) proportion (percentage) of the pixel intensity of human chromosomes 1, 5, 11, 13, and 17 in the two most peripheral and two most internal nuclear shells; n = 100 cells. The results from the three-dimensional analysis (lower panels) recapitulated those from the two-dimensional analysis (upper panels). *P < 0.05 and **P < 0.01, comparing the position of the chromosome in the NET-transfected cells to the NLS-GFP transfected control using Kolmogorov-Smirnov (KS) tests. Statistics for all chromosomes are given in Additional file 1.
	Figure 6. Removal of the transmembrane segments reduces effects of NETs in chromosome repositioning. Soluble fragments of NETs from their largest predicted nucleoplasmic segment were fused to an NLS and stably expressed in HT1080 cells to determine if they could influence chromosome repositioning. (a) Schematic diagram of each NET highlighting the soluble fragment in blue. The dark grey boxes indicate predicted transmembrane spans. (b) Images showing nucleoplasmic targeting of the GFP fusion constructs. (c) Cells expressing soluble fragments encoding the principal nucleoplasmic regions of NETs did not recapitulate the strong effects in repositioning observed with full-length NETs (Figure 5b). Error bars indicate standard deviation between the means of individual experiments. *P < 0.05 comparing the position of the chromosome in the NET-transfected cells to the NLS-GFP transfected control using KS tests. None of the soluble NET fragments yielded higher stringency P-values < 0.01. Statistics are given in Additional file 1.
	Figure 7. Reversibility of chromosome repositioning effects. (a) Wild-type HT1080 cells were separately treated with small interfering RNA (siRNA) oligos to deplete NET29 or NET39. Western blots of control siRNA and targeting siRNA-treated cells are shown. The signal intensity of bands on the blots was quantified and the band intensity for each siRNA was set to 1 for day 0. The normalized intensity of the bands over the time course is shown above each band. (b) Chromosome 13 position was quantified. Depletion of either NET reduced the endogenous peripheral positioning of chromosome 13. Error bars indicate standard deviation between the means of individual experiments. *P < 0.05 and **P < 0.01, comparing the position of the chromosome in the si NET cells to the si scramble control using KS tests. Statistics are given in Additional file 1.
	Figure 8. NETs that recruit chromosomes to the nuclear periphery have restricted tissue expression. Human tissue blots equally loaded for the tissues listed (20 μg in each lane) were probed with NET antibodies. NETs that had strong chromosome repositioning effects tended to be highly restricted in their expression. In contrast, several other NE proteins and controls were expressed in all tissues (lower panels). Ab1 and Ab2: two commercial antibodies to NET39 revealed restricted tissue expression and the highest levels of expression in muscle, but differed in the reactivity with some other tissues. Asterisk: NET47 migrates at 38 kDa instead of the calculated 46 kDa.
	Figure 9. Peripheral chromosome 5 position in liver is reduced by depletion of liver NETs. (a) Chromosome 5 in human liver or kidney sections. Scale bar, 5 μm. (b) Chromosome 5 positioning was quantified for the two tissues (n = 100). (c) NET45 and NET47 were depleted by RNA interference in HepG2 cells, a liver-derived human cell line. A scrambled siRNA sequence was used as a control. The knockdown over time is shown by western blot. The signal intensity of bands on the blots was quantified and the band intensity for each siRNA was set to 1 for day 0. The normalized intensity of the bands over the time course is shown above each band. (d) Chromosome 5 in siRNA-treated HepG2 cells (left, scramble control; right, NET depleted). Scale bar, 5 μm. (e) Quantification of the results in (d); 100 cells were counted for each timepoint. The percentage of chromosomes relocated away from the periphery by the combined NET45/NET47 knockdown increased over time, also strengthening the statistical significance. (f) NET45 and NET47 were also tested individually to determine if both were needed for the normal peripheral localization. Each alone reduced the percentage of chromosome 5 at the periphery. The combined knockdown had a stronger effect than either NET alone (n = 100). Error bars indicate standard deviation between the means of individual experiments. *P < 0.05 and **P < 0.01, comparing the position of the chromosome in the si NET cells to the si scramble control using KS tests. Statistics are given in Additional file 1.

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